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Every year, a significant amount of saffron petals remains as a by-product after harvesting and separating the thread-like parts of the flower (stigmas). The saffron stigma constitutes only 7.4% of the flower's weight and the remaining 92.6% is unused. This plant residue (petals) is a valuable source of plant bioactive compounds (phytobiotics), which based on previous studies, has useful effects on rumen fermentation, digestion and diet efficiency. However, there is no specific information about the effect of this by-product on ruminal microbial populations, enzymatic activity, antioxidant health, as well as the mechanism of effectiveness. In the present study, it was hypothesized that the use of low levels of saffron petals in the diet (as a natural additive) could improve microorganism populations, hydrolytic enzymes and antioxidant health of the rumen. Therefore, this research was carried out to investigate the mechanism of the positive effects of saffron petals on the energy and nitrogen metabolism and antioxidant health of the sheep rumen using the in vitro method.  Experimental treatments included a diet without saffron petals (control) and diets containing 1, 2, and 3% of saffron petals (based on dry matter). The 24 and 72-h gas production tests were performed in 2 series (runs; in different weeks) and 3 replicates in each run (i.e., 6 observations per treatment at each incubation time). The gas production from the samples incubated in the 100-mL glass syringes was recorded. Then, the protozoa (using a hemocytometer and light microscope), cellulolytic and proteolytic bacteria (using liquid medium and Hungate tubes), hydrolytic enzymes (via chemical methods and photometry), methane (by injecting NaOH solution to absorb CO2) and antioxidant capacity (via ferric reducing antioxidant power assay) were measured. Truly degraded substrate was determined by boiling fermentation residues in the neutral detergent solution and weighing, ammonia-N by phenol-hypochlorite method, and the volatile fatty acids (VFA) using gas chromatography. Microbial biomass production, digestibility, metabolizable energy and partitioning factor were estimated using the equations. Data were analyzed using the Proc GLM of SAS 9.1 in a completely randomized design (6 observations per treatment).  The inclusion of different levels of saffron petals in the diet decreased the in vitro ruminal protozoa population (P<0.05), due to its bioactive components, which damage the structure and function of the protozoa membrane, deactivate protozoa enzymes or deprive protozoa of the substrate and metal ions needed for their metabolism. Moreover, saffron petals increased the cellulolytic bacteria numbers and microbial biomass production (P<0.05). This was due to the reduction of protozoa, which leads to the reduction of the bacteria predation and thus improves the growth and population of the cellulolytics. The ruminal fibrolytic enzymes and amylase activity decreased with the use of saffron petals in the diet (P<0.05), because of the higher cellulolytic bacteria and microbial biomass, i.e., higher bacteria activity. These improvements resulted in more (P<0.05) diet digestibility, metabolizable energy, total VFA and truly degraded substrate in the saffron petals-containing groups. The inclusion of saffron petals in the diet resulted in decreasing in vitro ruminal methane release, ammonia-N production and acetate-to-propionate ratio (P<0.05). These changes were due to the decreased protozoa numbers leading to less hydrogen supply for methanogens and higher hydrogen shift toward propionate, as well as less deamination process and ruminal nitrogen recycling. Another reason was the increase of cellulolytic bacteria that consume ammonia as their main nitrogen source. Antioxidant capacity increased with the use of saffron petals in the diet (P<0.05), due to the bioactive compounds and antioxidants (such as flavonoids) present in this plant residue. The maximum truly degraded substrate was observed in the diet containing 3% of saffron petals. The lowest methane, protozoa and ammonia were in diets containing 2 and 3% of saffron petals. Moreover, adding 2 and 3% of saffron petals caused the highest antioxidant power.  The results showed that the positive effect of saffron petals on the ruminal metabolism and antioxidative health of sheep, in vitro, was due to the beneficial changes that occurred in microbial populations (increase of cellulolytic bacteria and decrease of protozoa), hydrolytic enzymes (fibrolytic enzymes and amylase) and its antioxidants. Therefore, low levels (up to 3% of diet) of saffron petals can be used as a natural phytobiotic additive to improve rumen fermentation and reduce loss of energy and nitrogen resources, although it is better to confirm the results in vivo

This study aimed to determine the chemical composition, silage characteristics, digestibility, and in vitro gas production parameters of saffron waste (including petals and stamen) before and after ensiling. Experimental treatments (4 replicates) contained: 1) Saffron wastes before ensiling: SWBE; 2) Saffron wastes after ensiling: SWAE; 3) 96.88% Saffron wastes after ensiling+3.12% wheat bran: 96.88%SWAE+3.12%WB (fresh weight); 4) 93.75% Saffron wastes after ensiling+6.25% wheat bran: 93.75%SWAE+6.25%WB (fresh weight); 5) 87.5% Saffron wastes after ensiling+12.5% wheat bran: 87.5%SWAE+12.5%WB (fresh weight); 6) 75% Saffron wastes after ensiling+25% wheat bran: 75%SWAE+25%WB (fresh weight); 7) 50% Saffron wastes after ensiling+50% wheat bran: 50%SWAE+50%WB (fresh weight). Statistical analysis of the data was performed in a completely randomized design. Treatment 2 did not have good quality and odor due to high moldy and adhesion characteristics, but the addition of wheat bran, especially at higher levels (treatment 4, 5, 6) led to an increase in the silage quality. Chemical composition of the experimental treatments (dry matter 10.40-54.37% of fresh weight, neutral detergent fiber 12.83-27.35%, acid detergent fiber 7.23.11.45%, crude protein 14.88-15.67%, ether extract 5.43-5.77% and ash was 5.89-11.12% of dry weight) was different. Among the experimental treatments, the highest neutral and acid detergent fibers were observed (27.35% and 11.45%, respectively) in treatment 7. The lowest pH as well as the highest concentrations of lactic and acetic acids, gas production at 12 and 24 h incubation, and constant rate of gas production were observed in treatment 7. True dry matter digestibility differed from 76.30% for treatment 2 to 79.95% for treatment 1. Overall, saffron waste had good nutritional value before being ensilaged, and ensiling without additives reduced its quality. However, it was possible to ensilage saffron wastes with wheat bran as a moisture-absorbing material without adverse effects on some nutritional parameters. In terms of appearance quality, odor, non-molding, and fermentation characteristics of the silo environment, the best conditions were observed in treatment 7.