C. Villani

Membrane integrity evaluation in rabbit spermatozoa

OBJECTIVES The behaviour of head and tail sperm membrane subjected to different vitality tests was evaluated, searching for possible correlations with sperm motility. STUDY DESIGN On fresh semen from seven rabbits we performed: the Eosin Exclusion Test (EET) and, with different incubation times (T(0), T(5), T(30)), a Hypoosmotic Swelling Test (HOS-test) and a combined Hypoosmotic and Eosin Test (HE-test). Kinetic parameters were determined by using the CASA system CELL-TRAK. Statistical analysis was performed by one way ANOVA and Pearsons correlation coefficient. RESULTS The HOS-test revealed significant differences between percentages of curled spermatozoa at T(0) (67.86+/-4.28) versus T(5) (72.86+/-3.28) and versus T(30) (75.14+/-6.5). Among four types of spermatozoa obtained with the HE-test, percentage of Type 1 sperm (HOS+/Eosine) at T(0) (32.3%+/-6.65), was lower than motile sperm (66.86%+/-9.72) but positively correlated. CONCLUSIONS The HE-test is a rapid instrument for predicting sperm motility, selecting spermatozoa with a high degree of membrane resistance both in the tail and the head compartment.

Assessment of trans, trans-muconic acid in human seminal plasma.

Summary. Trans, trans‐muconic acid (tt‐MA) is one of the most important metabolites of benzene, a pollutant ubiquitously distributed in ambient air and classified in 1982 as a group I carcinogen. For its sensitivity and specificity, tt‐MA excreted in urine is considered a good biological marker of benzene exposure. In this study, seminal tt‐MA levels in occupationally nonexposed subjects (n = 32) have been determined. The seminal fluid of normozoospermic subjects contained an average tt‐MA concentration (170 ± 100 ng ml−1) significantly lower than that of teratozoospermic (310 ± 180 ng ml−1; P < 0.01), oligozoospermic (400 ± 180 ng ml−1; P < 0.001), and oligoasthenozoospermic (430 ± 230 ng ml−1; P < 0.01) subjects. A negative correlation existed between tt‐MA levels and sperm concentration (r = − 0.62; P < 0.001), percentage of normal spermatozoa (r = − 0.41; P < 0.05), and percentage of vital spermatozoa (r = − 0.89; P < 0.001). Average tt‐MA levels detected in seminal plasma were higher in smokers (350 ± 160 ng ml−1) than in nonsmokers (280 ± 210 ng ml−1). These results show that seminal plasma tt‐MA content could be an important biological indicator for evaluating the negative effects of benzene on spermatogenesis.

A SOURCE OF FALSE-NEGATIVE RESULTS IN CLENBUTEROL ANALYSIS IN TISSUES OF VEAL CALVES

The possibility of false-negative results in clenbuterol analysis was investigated in bovine tissues. An extraction procedure currently in use was adapted to process 100 specimens of different tissues each time. Its efficiency and accuracy were investigated radiometrically by means of a series of different molar concentration of the tritiated drug. In samples not submitted to extensive delipidation, unreliability of the analysis was evident. The measurement of tissue clenbuterol content, by a competitive ELISA, gave results numerically similar to those existing in literature, but with an accuracy high enough to minimize the frequency of false-negative results.

Shared reaction in solid-phase immunoassay for estriol determination

In the search of factors responsible for the experimental difficulties in developing accurate and sensitive solid-phase immunoassay of steroids, an experimental model has been set up for the study of nonspecific interaction of the steroid analyte with the coating protein. Along with the development of a highly sensitive enzyme-linked, solid-phase immunoassay for estriol measurement, we observed evidence of shared reactions. This property, to our knowledge not previously described for monomeric, low-molecular-weight antigens like estrogens, has been attributed to the presence of bovine serum albumin, which is capable of binding estrogens through hydrophobic interactions. The addition of estriol in solution in large excess did not reach a complete inhibition of the binding, so the possibility was excluded that the antibody simply binds to the adsorbed estrogen. The simplest explanation for the occurrence of the reaction is the hypothesis that a family of antigen determinants arises when the estriol is conjugated to a protein carrier. The corresponding antibodies are revealed only when the estrogen participates to the actual analytical system in the form of a steroid-protein conjugate. In the experiment, the estriol has been recognized as being coupled with one or more amino acid side chains present around its site of covalent linkage to the immunogen protein. The discussed results may be of help in developing a solid-phase immunoassay of small antigens as steroids, but also in applying the hybridoma and phage display technologies, the screening methods of which are based on sensitized solid phases.