281 EVALUATION OF IN VITRO EMBRYO PRODUCTION EFFICIENCY USING SEXED BULL SPERM SORTED WITH TWO TYPES OF CELL SORTER

Abstract

Cell sorting is an important part of the sperm sexing process. The objective of this study was to compare the efficiency of in vitro embryo production using sexed frozen–thawed bull sperm sorted with 2 types of cell sorter. Ejaculates from 2 Bos taurus (Holstein, 5 years old) bulls underwent conventional processing (control) or sorting for X chromosome bearing sperm using MoFlo^® SX (SX, Dako, Fort Collins, CO, USA) or MoFlo^® XDP-SX (XDP, Beckman Coulter, Fullerton, CA, USA) following XY™ sperm-sorting protocols. Processed sperm samples were cryopreserved in 0.5-mL plastic straws. Cumulus–oocyte complexes obtained from abattoir-derived ovaries were matured for 20 h in HEPES–TCM-199 (Lu and Seidel 2004 Theriogenology 62, 819–830) and randomly assigned to each of 3 sperm treatment groups. Thawed sperm were centrifuged for 20 min at 448 × g through an ISolate^® (Irvine Scientific, Santa Ana, CA, USA) gradient (45:90%). Sperm pellets were washed in IVF100 (Hoshi 2003 Theriogenology 59, 675–685) by centrifugation for 5 min at 252 × g. Oocytes were co-incubated with washed sperm (5 to 10 × 10⁶ sperm mL^–1) in IVF100 (Hoshi 2003 Theriogenology 59, 675–685) for 8 h at 38.5°C in 5% CO₂ and 95% air (Day 0). Presumptive zygotes were cultured for 90 h in CDM-1 (Lu and Seidel 2004 Theriogenology 62, 819–830) and then washed and cultured in IVD101 (Hoshi 2003 Theriogenology 59, 675–685) at 38.5°C in 5% CO₂, 5% O₂, and 90% N₂. Cleavage rates on Day 2 and blastocyst rates on Day 7 to 9 were recorded after insemination. Two-way ANOVA was used for data analysis, followed by Fisher’s PLSD test. Experiments were replicated 4 times for bull A (total of 1 350 oocytes used) and 5 times for bull B (total of 1 529 oocytes used). The data are summarised in Table 1. No interaction was observed between the treatments and bulls. Cleavage rates were not significantly different in the 3 treatment groups. However, blastocyst rates were significantly lower in both SX (P < 0.001) and XDP (P < 0.002) groups than in control groups for both bulls but not different between SX and XDP (P > 0.8). Bull B showed significantly poorer results than bull A regarding both cleavage (P < 0.003) and blastocyst (P < 0.02) rates. MoFlo^® SX (analogue processing) has been used for a decade, and XDP (digital processing) is the replacement model with its accelerated sorting speed. The current results indicated that the in vitro embryo production efficiency did not differ between sperm sorted with either SX or XDP. We suggest that sperm can be sorted using XDP without compromising sperm health.

Table 1.  Cleavage and blastocyst rates after IVF with 2 Holstein bulls for three sperm treatments