Nano Se was prepared using sodium selenite and ascorbic acid according to the modified method of Qian Le et al. (2010). Finally, the gray precipitant obtained was filtered and washed by water and ethanol, respectively.

When the particle size is reduced to nano size, the properties of the materials are likely to be far different from the bulk materials. In the present study, Transmission electron microscopy, particle size analyser, and inductively coupled plasma mass spectrometry were used to analyse the properties like morphology, particle size distribution etc.

In vitro cytotoxicity assay was carried out in vero cell lines to ensure the safety of nano particle source of selenium as per the method of Mosmann, 1983. The parameters like per cent cell inhibition exhibited under different concentration of nano forms of selenium was studied.

The rumen liquor needed for the experiment was collected in slaughter house from six sheep immediately prior to slaughter and brought to the laboratory by maintaining the temperature of rumen liquor at 39 ºC and under anaerobic condition during the transit. Ruminal fluid was filtered through 4 layers of muslin cloth and stored in pre warmed thermos container at 39°C till its use.

The basal diet used for the experiment had roughage: concentrate in the ratio of 60:40. Roughage used in this study was Bajra Napier hybrid grass (CO₄). The concentrate mixture had maize (48.8 per cent), Soya bean oilcake (21.6 per cent) de oiled rice bran (25.6 per cent), salt (1.6 per cent) and calcite (2.4 per cent). The roughage component was ground to pass through 1.0 mm sieve. The roughage: concentrate mixture was well mixed to form the basal diet. There is also addition of sodium selenite, nano selenium and selenocysteine at different levels.

The accurately weighed individual feed ingredients were subjected to in vitro gas production studies. 500 mg of feed in 40 ml of buffered rumen fluid were incubated in prewarmed (40^oC) 100 ml calibrated glass syringes placed in an incubator as described by Menke et al. (1979).

An incubation period of 24 h was adopted for this study. At the end of the incubation, in vitro apparent dry matter digestibility (IVADMD), in vitro true dry matter digestibility (IVTDMD) and microbial biomass production were determined.

In vitro apparent digestibility of dry matter (IVADM) was determined as per Blummel et al. (1997_b). True digestibility was calculated as the weight of substrate incubated minus the weight of the residue after neutral detergent solution treatment.

The microbial biomass was calculated using the equation quoted by Blummel et al. (1997_a).

Microbial biomass = Substrate truly digested – Substrate apparently digested

The size and zeta potential of nano particle synthesized from sodium selenite is 31.8 ± 2.31 nm and−33.1 ± 6.5 mV respectively while product yield is 40.97%. The synthesized nano selenium powder contains 98.62% selenium, free from impurities, nano in nature and spherical in shape. Result is presented in table 1.

Similarly, Zhang et al. (2017) synthesized nano selenium with mean particle size of 36.8±4.1 nm using beta lactoglobulin as a stabilizer in redox system of ascorbic acid and selenite. Gangadoo et al. (2017) use solution phase synthesis approach for selenium nanoparticles by reducing selenium tetrachloride in the presence of ascorbic acid. Nanoparticle spherical in shape and have size 46 nm.

In order to determine safe level of inclusion of nano selenium, in vitro cytotoxicity test was done on in vero cell line with concentrations, 0.25, 0.5, 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 µg/ml for nano selenium. Based on the calculations, IC₅₀ for nano selenium derived from selenium powder was 89.11 µg/ml. The IC₅₀ is defined as the sample concentration that is required to reduce the absorbance to half that of the control and which would give the 50 % cell death. Since our inclusion level of nano selenium is 0.3 mg/kg and it revealed that the nano selenium is safe up to the level of 30 mg/kg to use as feed supplement as source of selenium in lamb ration.

Hashem et al. (2021) showed that IC50 of mycosynthesized Se-NPs was 316.73 μg/ml towards Vero cell line CCL-81. Salem et al. (2020) showed that IC₅₀ value of selenium nanoparticle against two different cell cultures, namely; human normal lung fibroblast (Wi 38) and human cancer colorectal adenocarcinoma epithelial (Caco-2) was 171.8 and 104.3 μg/ml respectively,

The results of the in vitro studies to assess effect of different forms of selenium on in vitro dry matter degradability (IVDMD) are presented in the table 2. From the results it can be inferred that when selenium is added at graded level to the basal diet in any form (inorganic, organic and nano) resulted in significant increase (p<0.05) in digestibility parameter such as in vitro apparent dry matter digestibility, in vitro true dry matter digestibility and microbial biomass production at all levels of addition compared to when no selenium was added. Rumen fermentation is a fundamental and special process of the ruminant metabolism and it determines the assimilation and intake of nutrients from ingested food.

In this trial, Inorganic, Organic and Nano selenium at graded levels when added to basal diet significantly (p<0.05) increased in vitro apparent dry matter digestibility, in vitro true dry matter digestibility and microbial biomass production at all levels of addition compared to unsupplemented group.

Consistent with our result Shi et al. (2011) stated that addition of nano selenium to the basal diet of sheep significantly (p<0.001) increased the feed utilization and rumen fermentation pattern through improvement in nutrient digestibility, whereas Ibrahim et al. (2018) reported that Nano selenium supplementation at 0.3 mg/ kg level improved nutrient digestibility, feeding value and growth performance in ossimi lambs compared to Sodium selenite and Selenium yeast form of supplementation.