Oral administration of Omega-3 affects post-thaw motility of canine spermatozoa

Sperm membranes are key structures that may affect semen quality and therefore its fertility and freezability. Membranes performance is closely related to the polyunsaturated fatty acid (PUFA) composition of their phospholipids, which become more prone to oxidation during the freezing-thawing process. In men, supplementation with Omega-3 in the diet improved sperm count [1]. The aim of this study was to evaluate whether a dietary supplementation with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may improve dog semen characteristics before cryopreservation and post-thaw. Semen was collected from 5 healthy, fertile dogs (four Labrador and one Golden Retriever, two to seven years old) on Days 0, 30, and 60 of an oral supplementation with 30 mg/kg of EPA and 20 mg/kg of DPA (Omega-3 Select, Jamieson, Ontario). Fractionated semen was collected by manual stimulation and evaluated for volume, concentration (by Thoma counting chamber) and subjective motility. Smears were prepared and stained with Spermac stain® (Minitube GmbH, Tiefenbach, Germany) to be evaluated counting 200 spermatozoa at 1000x. Ejaculates were centrifuged at 700g for 6 minutes, the supernatant removed and the sperm pellet diluted in two steps in a Tris, citrate and fructose extender with a final concentration of 5% glycerol and 0.5% Equex STM paste [2, modified]. Final sperm concentration was 100x10^6 spermatozoa/ml. After equilibration, semen was frozen placing the straws 4 cm above liquid nitrogen (LN) for 10 min before plunging them in LN. After being stored for at least one week, straws were evaluated for motility by a Computer Assisted Sperm Analyser (Ceros 12.1, Hamilton Thorne Inc., Beverly, USA) immediately post-thaw and after 1 and 2 hours (T0, T1 and T2, respectively) of incubation at 37°C. Data were analysed by ANOVA including in the model the effect of dog and time (Day 0, Day 30, Day 60). There was neither an effect of dog nor of time on fresh semen characteristics, except for the proportion of morphologically normal sperm cells, that was affected by dog. Tail defects increased significantly between Day 30 and Day 60 (from 2.9±0.5% to 6.3±1.7%, P<0.05). Between Day 0 and Day 30 of supplementation, a significant improvement was observed for total motility (at T1 and T2), progressive motility (at T2) and rapid spermatozoa (at T2). Post-thaw total motility, however, significantly decreased between Day 30 and Day 60 (at T0: from 64.1% to 50.6%, at T1: from 57.6% to 24.3% and at T2: from 54.0% to 13.1%, respectively). A similar statistically significant decrease between Day 30 and Day 60 was observed for progressive and rapid motility. It thus appear that after an initial improvement in semen freezability, administration of Omega-3 for 60 days has a deleterious effect on semen characteristics of normal dogs. It can be hypothesized that an elevated uptake of PUFA in sperm membranes makes the endogenous antioxidant activity non efficient. It should be evaluated if a contemporary administration of antioxidants may prevent the negative effects observed at Day 60. [1] Safarinejad MR. Andrologia 2011; 43:38–47. [2] Rota A, et al. Theriogenology 1997, 47: 1093-1101.