159 EFFECT OF GRANULOCYTE-MACROPHAGE COLONY STIMULATING FACTOR (GM-CSF) AND SERUM, ALONE OR IN COMBINATION, ON INTERFERON-TAU (IFNT) PRODUCTION BY OVINE EMBRYOS

Abstract

The objectives were to establish whether GM-CSF, which may stimulate IFNT production by blastocysts at implantation, influences IFNT production by ovine embryos in culture and whether GM-CSF can explain stimulation of IFNT production by ovine serum (Rooke et al., 2003 Reprod. Abstr. Series 30 : 56). Oocytes, aspirated from sheep ovaries obtained from a local abattoir on 4 separate days, were matured and fertilized by standard procedures. On Day 1, cleaved zygotes (approximately 10 per 0.05-mL⁻¹ drop; total, 175 per treatment) were cultured in synthetic oviductal fluid containing either 0.3% bovine serum albumin and amino acids (SOFA) or 10% sheep serum (SOFS) in the presence (+) or absence (−) of 5 ng mL⁻¹ ovine GM-CSF. Medium was changed every 2 days. On Days 6 and 7, blastocysts were removed from their group drops and assigned in a balanced manner individually to 0.05-mL⁻¹ drops of one of the 4 media for a further 24 h. Embryo grade, diameter and cell number were recorded. IFNT concentrations in conditioned media samples were determined by ELISA. The presence of serum accelerated blastocyst development (cross-tabulation; chi-square analysis; P = 0.039; Table 1). Both serum and GM-CSF in group culture media increased (2 × 2 factorial ANOVA, P < 0.001) IFNT concentrations (ng mL⁻¹; SOFA−, 0.5; SOFA+, 1.7; SOFS−, 2.7; SOFS+, 4.2; SED, 0.47) with no interaction between serum and GM-CSF. After 24 h individual culture of Day 6 blastocysts, IFNT concentrations (2 × 2 factorial ANOVA; Table 1) were influenced mainly by the origin of the blastocyst (group treatment covariate, serum, P < 0.001; GM-CSF, P < 0.001) rather than by individual blastocyst culture media (GM-CSF, NS; serum, P < 0.001). The pattern of results was similar for Day 7 blastocysts. Incubation of zygotes in the presence of GM-CSF had no effect on embryo grade or diameter but blastocysts (both day 6 and 7) incubated individually for 24 h in the presence of GM-CSF had more cells (128 v. 119; SED, 3.52; P = 0.001) than blastocysts incubated concurrently without GM-CSF. However, the pattern of IFNT production after correction for cell number was unchanged. In conclusion, culture of zygotes in medium containing GM-CSF or serum, alone or in combination, from Days 1 to 7 increased IFNT production, and IFNT production by individually cultured blastocysts depended on their origin. SAC receives financial support from the Scottish Executive Environment and Rural Affairs Department.

Table 1 
Blastocyst yields (% of zygotes) on Days 6 and 7 and IFNT concentrations (ng ML⁻¹) in media after individual culture of Day 6 blastocysts