Effect of L-glutamine for freezing equine embryos: evaluation by DAPI staining and transfer of multiple embryos to...

Day 6.5 equine embryos (n=30) were frozen in a medium containing glycerol (2.5-10.0%) supplemented with 0, 20 or 100 mmol L-glutamine 1(-1). After thawing, the embryos were tested individually, using 4',6'-diamidino-2-phenylindole (DAPI) staining to evaluate cell death. Three embryos (one frozen at each L-glutamine concentration) were transferred together into individual recipient mares. Pregnancy diagnosis was performed at day 12 (age of embryo). Embryos were collected at day 14 (age of embryo) and were identified by PCR amplified microsatellite analysis. Nine of ten recipient mares that received multiple embryos were pregnant: one mare was pregnant with two embryos and the other eight mares were pregnant with one embryo. The pregnancy rate was significantly higher (P < 0.05) for embryos transferred to recipient mares after freezing in media containing 100 mmol L-glutamine l(-1) (6 of 10 pregnancies) than for embryos frozen in media containing either 20 or 0 mmol L-glutamine l(-1) (1 of 10 and 3 of 10 pregnancies, respectively). Furthermore, DAPI staining analysis indicated that the percentage fluorescence was significantly lower (P < 0.05) in embryos frozen in media containing 100 mmol L-glutamine l(-1) (4.6 +/- 2.4, n=10) than in embryos frozen in media containing either 20 or 0 mmol L-glutamine l(-1) (23.0 +/- 31.3 and 10.8 +/- 9.5, n=10, respectively).