Water content of hooves seems to be one of the most determinant factors in hoof softness that may result in higher susceptibility to digital lesions. Formalin and copper sulfate known as their effects on hoof hardening in hoof bathes. In this current study in vitro effect of formalin and copper sulfate on hoof water content was investigated. Forelimbs of six male (6 months to two years old) Holstein-Friesian cows were cut in slaughterhouse and transferred to lab. Hooves of the left forelimbs used as treatment group, and right forelimbs used as control positive and negative . three samples, toe (T), sole (S) and Heel (H) were cut using surgical trephine (8mm). samples of Medial claws of the treatment group soaked in copper sulfate 5% (Cu) solution for 30 minutes and the samples of lateral claws soaked in formaldehyde 5% (Fld) solution for 30 minutes. Tap water used as positive control and in negative control group claws didn’t receive any treatment. Samples were weighted and incubated in 102 degree oven to get dry and each 24 hours weighted again until the difference between two measurements reduce to 0.04 grams. Weights were corrected in according to the smallest sample. Statistical analysis was done by three way ANOVA, and p values less than 0.05 revealed as significant.

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