Anticoagulants, such as ethylenediaminetetraacetic acid (EDTA), sodium citrate (Na-citrate), or heparin are normally used in hematological clinical tests to prevent coagulation. Although anticoagulants are fundamental for the correct application of clinical tests, they produce adverse effects in different fields, such as those involving specific molecular techniques; for instance, quantitative real time polymerase chain reactions (qPCR) and gene expression evaluation. For this reason, the aim of this study was to evaluate the expression of 14 genes in leukocytes that were isolated from the blood of Holstein cows, and which were collected in Li-heparin, K-EDTA, or Na-citrate tubes; then, they were analyzed using qPCR. Only the SDHA gene showed a significant dependence (p ≤ 0.05) on the anticoagulant that was used with the lowest expression; this was observed in Na-Citrate after being compared with Li-heparin and K-EDTA (p < 0.05). Although a variation in transcript abundance with the three anticoagulants was observed in almost all the investigated genes, the relative abundance levels were not statistically significant. In conclusion, the qPCR results were not influenced by the presence of the anticoagulant; thus, we had the opportunity to choose the test tube that was used in the experiment without interfering effects impacting the gene expression levels caused by the anticoagulant.

Graphical Abstract Summary: For its fat and protein contents, whole cottonseed (WCS) may be defined as a concentrate. On the other hand, its fiber is similar to that of forages in terms of effectiveness in the rumen. Considering the high digestibility of WCS fiber, its inclusion in calf starter could improve growth and rumen development. Hence, we investigated the effect of including WCS at 8% on growth performance, inflammometabolic profile, and rumen behavior in Holstein heifer calves. Calves receiving WCS in the starter showed increased average body weight (BW), improved metabolic profile, and longer rumination time compared with the control group. These results suggest that WCS may be included as a starter ingredient with beneficial effects.