The objective of this study was to evaluate the effect of using pistachio (Pistachio vera) hulls (PH) as a replacement for alfalfa hay (AH) in the diet of Baloochi sheep on their rumen microbial and rumen fermentation characteristics. Six Baloochi sheep with a body weight of 40.1 ± 1.77 kg (mean ± SEM) fitted with rumen cannulae were assigned at random to three diets in a double 3 × 3 Latin square design. The dietary treatments were control (basal diet), low PH (LPH) diet (0.50 of AH in basal diet replaced by PH), and high PH (HPH) diet (all of the AH in the basal diet replaced by PH). The daily basal diet was 400 g AH dry matter (DM), 200 g wheat straw DM, 168 g barley grain DM, 24 g cotton seed meal DM, 6.4 g vitamin–mineral supplement DM and 1.6 g salt DM. Bacterial populations were assessed by DNA extraction of samples of rumen liquor followed by real-time polymerase chain reaction analysis. Rumen samples were evaluated for pH, volatile fatty acid (VFA) and ammonia nitrogen (AN) concentrations. The populations of total bacteria (ng per l extracted DNA – control 564 versus LPH 407 versus HPH 315), Fibrobacter succinogenes and Ruminococcus albus decreased significantly as the level of PH in the diet increased (P < 0.05). The population of Ruminococcus flavefaciens in the rumen liquor was not affected by the LPH diet but was reduced to approximately half the level of the control in the HPH diet (NS). The replacement of AH partly or wholly in the diet caused an increase of rumen pH from 6.03 for control to 6.35 for LPH and 6.33 for HPH (P < 0.01). Inclusion of PH in the diet reduced total VFA concentration (control 95.0, LPH 76.3 and HPH 76.5 mmol/L; P < 0.01), individual VFA concentration (e.g. acetate control 47.6, LPH 35.1 and HPH 31.7 mmol/L; P < 0.01), and AN (control 193, LPH 173 and HPH 84.3 mg/L; P < 0.01) in the rumen liquor. It was concluded that the tannins in PH reduced the number of cellulolytic and total bacteria. Inclusion of PH in the diet of sheep also caused an increase in the pH and reduced the concentrations of AN and VFA of rumen liquor.

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