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Pima cotton production is increasing in the United States, but Pima cottonseed generally contains higher concentrations of the antinutritive pigment gossypol than conventional upland cottonseed. Heating promotes the reaction of gossypol with protein, reducing gossypol absorption and toxicity. The objective of this study was to assess the nutritional value for dairy cattle of Pima cottonseed cake (PCSC) that was heated and oil largely removed by an experimental extrusion process, compared with upland cottonseed (UCS) and Pima cottonseed (PCS). The PCS had greater crude protein (CP) and ether extract, less neutral detergent fiber (NDF) and acid detergent fiber (ADF), similar total gossypol, but higher (-)-gossypol isomer compared with UCS. Extrusion reduced lipid content by 73%, increased concentrations of CP, NDF, and ADF, and reduced total gossypol, (+)-gossypol, and (-)-gossypol in PCSC versus PCS. Forty lactating Holsteins (8 with ruminal cannulas) were blocked by days in milk into 5 squares in a replicated, incomplete 8 × 8 Latin square, and were fed diets containing, on a dry matter (DM) basis, 30% alfalfa silage, 31% corn silage, 21 to 25% high-moisture corn, and about 15% CP. Diets were fed as total mixed rations for ad libitum intake. Supplemental CP was from (1) solvent soybean meal (SSBM) only or 50% from SSBM plus 50% from (2) UCS, (3) PCS, (4) PCSC, (5) UCS plus PCS, and (6) UCS plus PCSC, or (7) 50% from expeller soybean meal (ESBM) plus 50% from PCS, and (8) 50% from ESBM plus 50% from PCSC. Periods were 4 wk long (total of 16 wk); production data were collected over the last 2 wk and blood and ruminal samples were taken on d 28 of each period. Data were analyzed using Proc Mixed of SAS (SAS Institute Inc., Cary, NC). Diet affected dry matter intake, with greatest intake on diet 6 and lowest intake on diets 1 and 3. The highest milk fat content was observed on diet 5 and the greatest fat yield on diet 7; fat content and yield were lowest on diet 1 (soybean meal control). Milk fat secretion was proportional to dietary fat content, indicating that cottonseed oil was used effectively for milk fat synthesis. We observed a trend for an effect on milk protein yield with the greatest protein secretion occurring on diet 7. Milk urea was lowest on diets 3, 7, and 8. Ruminal concentrations of branched-chain volatile fatty acids were lower, or tended to be lower, when PCSC replaced either UCS or PCS in the diet, suggesting reduced degradation and increased escape of PCSC protein. Among cottonseed-containing diets, total gossypol intake was lowest on PCSC, intermediate on PCS, and highest on UCS. Total gossypol and both (+)- and (-)-isomers of gossypol were higher in blood plasma on PCS and lower on PCSC than on the corresponding diets containing UCS, indicating that the extrusion process reduced gossypol absorption. In this trial, production on diets supplemented with UCS, PCS, or PCSC was comparable to that on diets containing soybean meal., (Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)