351 INSEMINATION OF HOLSTEIN HEIFERS WITH SEXED SPERM PROCESSED WITH OR WITHOUT EGG YOLK IN THE GLYCEROL-CONTAINING FREEZING MEDIUM

Abstract

Preparation of media for use in processing sorted sperm is laborious and expensive. The objective was to determine whether pregnancy rates in heifers following AI of frozen/thawed X chromosome-bearing sperm were equivalent for sperm processed for cryopreservation using glycerol-containing medium with or without egg yolk. X chromosome-bearing sperm from each of two Holstein bulls were isolated on the basis of DNA content using a flow cytometer. Sorted sperm were collected in 50-mL plastic tubes containing 2-mL of 20% egg yolk-TRIS extender without glycerol until each tube contained 12.4 mL and approximately 12 million sperm. Sorted sperm were cooled (5°C) over 90 min before the addition of 12% glycerol-containing TIS medium with or without 20% egg yolk, as two equal volumes (6.5 mL) at 15-min intervals. Tubes containing 25 mL were centrifuged at 850g for 20 min (5°C). After the supernatant was removed, sperm pellets within treatments and bulls were pooled. Sperm concentration was adjusted to 10 × 10⁶/mL with medium containing 6% glycerol and 20% egg yolk-TRIS. Sorted sperm were packaged and frozen in 0.25 mL-coded straws on freezing racks in LN₂ vapor. Straws from each bull-treatment combination were equally packaged in goblets to assure random use. Post-thaw sperm progressive motility, determined with time-lapse photography ('track motility'), was 44% for non-egg yolk and 43% for 20% egg yolk-glycerol-containing media. Nonsynchronized Holstein heifers (n = 119) were inseminated 4, 12, or 24 h after observed standing estrus. Sperm were deposited into the uterine body anterior to the internal cervical os. Approximately 1 month after insemination, pregnancy was determined using ultrasound. Data were subjected to ANOVA. The pregnancy rate and 95% confidence interval for sorted sperm processed with non-egg yolk-glycerol-containing medium was 56% (49-73; n = 59), and 55% (38-62; n = 60) for the 20% egg yolk-glycerol-containing medium (P > 0.1). Bull or inseminator effects on pregnancy rates were not significant (P > 0.01). Pregnancy rates as a function of time of insemination at 4, 12, or 24 h relative to observed estrus were 62% n = 65; 48% n = 48; and 50% n = 6, respectively (P > 0.1). Even though the scope of the field trial was limited, omission of egg yolk from the glycerol-containing medium did not adversely affect pregnancy rates. Preparation of non-egg yolk-glycerol-containing medium greatly reduces associated time, labor, and expense. This study also resulted in quite acceptable pregnancy rates with insemination doses of 2 × 10⁶ total sorted frozen/thawed sperm.