14 MOTILITY AND FERTILITY OF BULL SPERMATOZOA FROZEN IN EGG YOLK EXTENDER SUPPLEMENTED WITH LACTOFERRIN

Abstract

Lactoferrin (Lf) is secreted by the prostate, seminal vesicles, and epididymis, and appears to represent a major component of the sperm-coating antigens that protect sperm. The aim of this study was to determine the effects of Lf used in semen extender on motility and fertility of bovine frozen semen. Semen samples were collected from three fertile Japanese Black sires. The semen collected was cooled and extended with egg yolk–Tris extender supplemented with glycerol and Lf (from bovine milk) to give final concentrations of 7% and 0, 10, 100, 500, 1000, or 2000 µg mL^-1, respectively. After being extended, the semen was packaged in 0.5-mL straws, frozen, and transferred into liquid nitrogen. At least one week later, the frozen semen was thawed and washed mTALP containing caffeine. Motile sperm were separated by discontinuous 2-step (40 and 80%) Percoll gradient centrifugation. Sperm motility was calculated as the percentage of motile sperm separated by Percoll gradients relative to pre-separated sperm. Sperm motility character (motile sperm, progressive sperm) was analyzed by a CASA system (Hamilton Thorne Biosciences, Beverly, MA, USA). In the following experiment, we examined the effect of Lf added in the extender on improvement of semen fertility. The frozen semen from one sire extended with or without 500 µg mL^-1 of Lf was prepared (99 straws with Lf, 99 straws without), distributed to three inseminators, and used to inseminate to 198 cows. The sperm motility and motility character were analyzed by ANOVA and Fisher's exact test. The field trials were evaluated by chi-square test. After selection of motile sperm with the Percoll gradient, examination of sperm motility revealed that addition of Lf in the extender increased sperm motility significantly (P < 0.05) compared to that in the control without Lf: 51.2–53.8% in semen with added Lf (100–2000 µg mL^-1) vs. 39.6% in the control. CASA analysis showed that motile sperm and progressive sperm were improved significantly by the addition of Lf; the peak level was 500 µg mL^-1 of Lf in the extender. The percentage of motile sperm in semen with added Lf was higher significantly (P < 0.01) than that of the control: with Lf of 100 and 500 µg mL^-1, 50.8 and 56.0%, respectively, vs. 39.7% without Lf. The percentage of progressive sperm was also significantly higher (P < 0.05) than that of the control: with Lf of 100 and 500 µg mL^-1, 18.8 and 19.0%, respectively, vs. 13.5% without Lf. In the next experiment, we performed field trials to assess the availability of Lf for artificial insemination. In total, the group with Lf showed some but not a significant increase in fertility rate (delivery cow/inseminated cow) compared with the control group without Lf: 56.6% vs. 52.5%, respectively. In conclusion, these results suggest that addition of Lf in sperm extender may be useful for improvement of sperm motility and motility character after freezing and thawing. In the present study, we used fertile bulls, but the concentration of intact Lf in the semen was unknown, which might be the reason why the effect of Lf on the fertility rate was not observed to be significant. In future experiments it would be valuable to demonstrate the effect of Lf in subfertile semen.