238 ASSOCIATION BETWEEN SPERM MORPHOLOGY AND IN VITRO EMBRYO PRODUCTION IN MICE

Abstract

Although sperm morphology affects male fertility in several species, normally, it is not considered for in vitro fertilization (IVF) programs in mice. In order to correlate sperm morphology with IVF rates in murines, a total of 3336 oocytes were used in 11 identical IVF replicates using 8-wk-old B6D2 F1 (C57BL/6 × DBA/2J) males and 3- to 4-wk-old C57BL/6J donors females. For each replicate, 10 females were injected intraperitoneally with 5 IU of eCG (Novormon, Syntex, Buenos Aires, Argentina), and 5 IU of hCG (Ovusyn, Syntex) 48 h apart. Superovulated females were killed 14 to 16 h after hCG injection. The oviducts were isolated and cumulus–oocyte complexes were recovered and introduced into each sperm suspension drop. Previously, sperm was obtained from males killed by cervical dislocation, the cauda epididymis were recovered, minced with fine scissors in equilibrated human tubal fluid medium (HTF, EmbryoMax, Chemicon International, Phillipsburg, NJ, USA), and resuspended in a total volume of 200 μL of HTF. The sperm suspension was incubated at 0.05 or 3 × 10⁶ spermatozoa/mL concentration in equilibrated 100-μL drops of HTF under embryo tested mineral oil (Sigma, St. Louis, MO, USA), at 37°C in 5% CO₂, 95% air for 1 h before insemination. To evaluate sperm morphology, a sample of each replicate was fixed with 10% formalin and observed under phase-contrast microscopy (Olympus IX81) at 100× magnification. Sperm abnormalities regarding head and neck, tail, and cytoplasmic droplet were recorded from 200 cells. Five hours after insemination, the fertilized eggs were washed and transferred in groups of 50 to 100 into 100-μL drops of equilibrated culture medium (KSOM, EmbryoMax, Chemicon International) under the same culture conditions. The number of 2- (cleavage) and 8- (development) cell embryos was scored after 24 and 72 h in culture, respectively. Statistical analysis was performed by logistic regression taking into account the effect of the sperm abnormalities, the replicate, and the sperm dose. The effect of the sperm abnormalities is shown in Table 1. Results were not significantly influenced by the replicate or the sperm dose (P > 0.05). In conclusion, sperm morphology affects cleavage and embryo development rates in mice, and it should be taken into account as a source of variation in the success of IVF.

Table 1.  Effect of total sperm abnormalities on cleavage rate and embryo development in mice