Abstract
To provide new insights into the mechanisms through which seminal plasma proteins can protect sperm from damage caused during refrigeration, we evaluate the possibility that β-NGF can contribute to the improvement of sperm quality after cooling. First, β-NGF was detected in refrigerated sperm and compared with unrefrigerated sperm by western blotting of the proteins adsorbed by sperm, showing that native β-NGF is still present even 24 h after cooling only as an active form. Then, the effect of exogenous β-NGF on sperm quality after cooling was evaluated. A total of 12 ejaculates from male llamas (three ejaculates per male), were obtained by electro-ejaculation, diluted 4:1 with buffer Hepes-balanced salt solution and centrifuged at 800 × g for 8 min to remove the seminal plasma. Sperm were suspended in Tris-citrate-fructose-egg yolk diluent for a final concentration of 30 ×106/ml and cooled at 5°C for 24 h. After refrigeration, the extended sperm were equilibrated for 5 min at 37°C and divided into the following subgroups: sperm samples without treatment (control) and sperm samples supplemented with exogenous human β-NGF (10, 100, and 500 ng/ml). At 5, 30, and 60 min of incubation sperm were evaluated for sperm viability (using eosin/nigrosin stain), sperm motility and vigor (observed under light microscopy), and mitochondrial activity (using the JC-1 fluorescent marker). Vigor data were analyzed with the nonparametric Kruskal-Wallis test. The rest of the variables were analyzed with a mixed models approach. Mean comparisons were performed using Fisher's LSD test with a confidence level of 95%. A principal components analysis was performed to analyze the relationships between variables. Treatment of 24 h cooled sperm with 10 or 100 ng/ml of human β-NGF increased the percentage of total motility and vigor (p < 0.05). Besides, an incubation time of 60 min would be adequate to improve sperm quality, since all variables are positively related. The significant improvement observed in the motility and vigor of post-refrigerated sperm suggests that supplementation with exogenous β-NGF may be profitable for the improvement of cooled llama sperm.
Highlights
Artificial insemination (AI) is an important technique to ensure rapid genetic progress
When ejaculated and epididymal spermatozoa were analyzed, we found that β-NGF was present only in ejaculated sperm, indicating that this factor is provided by the seminal plasma during ejaculation [12]
The interaction between βNGF concentration and time was not significant indicating that the effect of β-NGF addition on sperm motility is independent of the incubation time
Summary
Artificial insemination (AI) is an important technique to ensure rapid genetic progress. Different protocols for sperm cooling and freezing, as well as semen extenders are currently under investigation. Higher pregnancy rates have been achieved using cooled sperm [3, 4] than with frozen-thawed semen [1, 5, 6]. A protocol for AI or cryopreservation of camelid semen should be designed taking into account the striking particularities of semen composition and spermatozoa. Camelids account for a higher concentration of β-NGF when compared with other mammals [10]. This factor is responsible for inducing ovulation in camelid females once it is deposited in the genital tract during copulation [11]. We provided some evidence that seminal β-NGF may have roles in the regulation of llama sperm physiology [12]
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