Evaluation of Chilled Dog Semen Extended With Sperm Activator

Abstract

Within modern biotechnology, different tools and methodologies have been developed to maximize canine semen conservation protocol to optimize reproductive results. In the last decades, the survival of chilled semen has been prolonged from 2 to 3 days with the first basic diluents, to 10-14 days with the modern extenders. However, their main limitation is that sperm quality decreases during cold storage. Sperm activators (SA) have been produced to provide the molecules necessary to maximize the sperm survival and quality with the aim to enhance fertility and prolificacy. In this study, the effect of commercial extender SA (Theriosolution® Canine AI extender -Chile-) was recorded by daily evaluation of chilled semen for 14 days. In this experiment, sperm-rich ejaculate fraction was collected from six adult healthy Neapolitan Mastiff dogs. The semen evaluation started immediately after collection (d0), and after that a next generation extender was added (d0) for every 24 h from d1 (with and without SA) to d14, to determine spermatozoa progressive motility, velocity of forward progression (VFP), morphology, and integrity of the spermatic membrane. The initial sperm concentration of extended semen was 417.3 ± 170.4 x 10⁶/mL (mean ± SEM) with 85.89 ± 4.76% of MNS (morphologically normal sperm), 84.47 ± 5.22 % live sperm, and pH of 6.2 ± 2.8. The initial VFP was 3.83 ± 0.48, but after 1 min with SA, it rises to 4.45 ± 0.45 (P < 0.001). The sperm progressive motility parameter increases significantly (P < 0.05) in experimental trial, respect to control, starting to d2 at finish (except for d7). The VFP analysis significantly increases in experimental trial (P < 0.05) during most days of the study with the exclusion of d3 and d14. To evaluate the seminal characteristics over time, the experiment was divided into T1 (d0-d5), T2 (d6-d10), and T3 (d11-d14) (P < 0.001) in evaluation of morphology and membrane functionality. The MNS reached 70% at d10 and finally 65% at d14, being considered normal and possibly fertile. With Host-s, 65% of MNS were also achieved at d14. The presence of glucose and fructose in the diluents used for refrigeration can exert very important effects given the fact that metabolic routes have been found in both sugars, providing both different and complementing effects. It can be concluded that the use of SA prior to artificial insemination improves the quality of chilled semen significantly, although it does not reverse the effects of deterioration due to cellular metabolism over time.

Keywords: andrology; assisted reproductive biotechnologies (ART); chilled semen; longevity; semen activator extender (SA); sperm cryopreservation; sperm evaluation.

Figure 1

Variation of sperm velocity of forward progression [according to Howard et al. (17)] after cooling and timing. Mean ± SEM *P < 0.05. The graphed evaluations correspond to 1 min after adding SA.

Figure 2

Progressive motility with and without the effect of seminal activator during the 14 d of the evaluation of refrigerated semen. Mean ± SEM *P < 0.05. The graphed evaluations correspond to 1 min after adding the SA.

Figure 3

Velocity of forward progression and sperm progressive motility on day 14 after the placement of the sperm activator (Mean ± SEM). The variation was calculated as the difference between each value (progressive motility and velocity of forward progression) of each day with the course of the evaluation time (1, 5, 10, and 15 min).

Figure 4

Temporal variation of sperm morphology by vital staining (Mean ± SEM). *Difference between of head or middle piece sperm defects and significant tail (P < 0.05).

Figure 5

Variability of semen membrane functionality (Host-s) of refrigerated samples.