Purification and Quantification of Lactoferrin in Equine Seminal Plasma.

Abstract

ABSTRACT. Lactoferrin with a molecular mass of 80 kDa was purified from equine seminal plasma by heparin-Agarose affinity chromatography and Sephacryl S-200 gel filtration. Purified lactoferrin was found to be highly homogeneous on the bases of its migration as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and of the monospecificity of rabbit antibodies to the purified protein in immunoblotting of seminal plasma proteins. A sandwich enzyme-linked immunosorbent assay was developed for quantifying lactoferrin in equine seminal plasma. Seminal plasma lactoferrin concentrations in 23 normal stallions ranged from 42 to 453 µg/ml, with a mean value of 157 ± 118 µg/ml (S.D.).

Seminal Plasma Lactoferrin but Not Transferrin Reflects Gonadal Function in Dogs

“…The molecular mass (75.2 kDa) of lactoferrin purified from canine seminal plasma in the present study is equivalent to that of lactoferrin purified from human (76 kDa) [23] and equine (80 kDa) [14] seminal plasma. It was recently found in swine that lactoferrin is first secreted as a 75-kDa glycoprotein but carbohydrate moieties are gradually digested resulting in a 70-kDa protein in the cauda epididymidis [16].…”

“…Equine lactoferrin and antiserum to equine lactoferrin: Equine seminal plasma lactoferrin and rabbit antiserum to equine seminal plasma lactoferrin were prepared as previously described [14].…”

“…Seminal properties including sperm number and sperm motility were immediately examined and the seminal samples were subsequently centrifuged at 1,650 × g for 20 min at room temperature. The seminal plasma was collected and stored at -25°C until use.Equine lactoferrin and antiserum to equine lactoferrin: Equine seminal plasma lactoferrin and rabbit antiserum to equine seminal plasma lactoferrin were prepared as previously described [14].Transferrin and antibody to transferrin: Highly purified canine serum transferrin was obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Affinity-purified goat anticanine transferrin antibody and alkaline phosphatase-conjugated goat anti-canine transferrin antibody were obtained from Bethyl Laboratories, Inc. (Montgomery, TX, U.S.A.).…”

“…65(6): 679-684, 2003 Lactoferrin is an iron-binding protein (molecular mass of 80 kDa) that is found in various exocrine fluids, blood plasma and specific granules of neutrophils [15]. Lactoferrin has been identified in the seminal plasma of humans [10,19], swine [21] and horses [14]. Lactoferrin seems to be synthesized mainly in the epididymis of mice [27], swine [16] and horses [7], and it is present in the prostate and seminal vesicles, but not in the epididymis, of humans [26].…”

Relationship between Seminal Plasma Lactoferrin and Gonadal Function in Horses

“…Lf found in substantial amounts in milk is considered to promote iron absorption in infants [7]. Lf has been found in the seminal plasma of various mammalian species [4,6,10,[13][14][15], and identified as the sperm-coating antigen [4,8,14]. It is known that Lf is synthesized in the epididymis in various mammalian species including horses [3], pigs [8], and mice [17].…”