Effects for fertility of processing steps of a new technology platform for producing sexed sperm

Abstract Text:

To understand the importance for fertility of key processing steps used in the production of sexed sperm, we conducted a series of field trials in U.S. dairy herds. Steps studied were: staining and processing in the absence of laser excitation (1), excitation laser power (2), and the presence of bisected sperm and debris resulting from laser-based cell destruction (3). For experiment 1, split or coincident collections from 8 dairy bulls were used to produce STAINED-L (2×10⁶ motile sperm/straw) and untreated controls (CON-H, 10×10⁶ sperm). The STAINED-L comprised sperm stained using Hoechst 33342 and subjected to all steps of the sexing process but with no exposure to laser excitation. Conception rates (CR) in Holstein heifers were 56.1%±2.8% (n=312 pregnancy diagnoses) for STAINED-L and 66.7%±2.6% (n=330) for CON-H. For experiment 2, one collection from each of 4 Holstein bulls was split to create untreated CON-L and stained treatments receiving low, medium, and high laser power during excitation. Post-thaw, these 4 treatments contained similar numbers of progressively motile sperm/straw (0.90 to 1.41 ×10⁶).  When used in virgin heifers, CR for all laser excited treatments were lower than CON-L (P<0.01), and were 58.7%±2.0% (n=591) for CON-L and 45.2%±3.6% (n=186), 44.9%±3.5% (n=205), and 39.1%±3.5% (n=192) for low, medium, and high laser excitation power, respectively. For experiment 3, TRT-L containing 3×10⁶ bisected sperm + 2×10⁶ unprocessed sperm was compared to matched untreated controls (CON-L, 2×10⁶ sperm), and contemporaneously produced high dosage controls (CON-H, 10×10⁶ sperm). The TRT-L was produced by processing an aliquot from each ejaculate through the laser detection + laser-destruction system set to bisect 90% of sperm and after each 1 hour of collection, combining equal portions of bisected and unprocessed spermatozoa. Post-thaw, matched low dosage fractions contained similar numbers of progressively motile sperm/straw (1.1 to 1.7 ×10⁶ for TRT-L and CON-L), but differed markedly in percentage of bisected sperm (69 to 79% and 32 to 59% non-motile, respectively). For CR in virgin heifers, TRT-L did not differ from CON-L but was lower than CON-H (P<0.01). Conception rates were 43.5%±1.9% (n=666) for TRT-L, 43.7%±1.9% (n=668) for CON-L, and 58.1%±1.4% (n=1334) for CON-H. Staining of sperm, excitation laser power, and sperm dosage have implications for the fertility of sexed sperm produced using this novel technology. Contrary to theory, the presence of significant numbers of non-motile and bisected spermatozoa and their debris did not impact conception rates.

Keywords:

conception rate, flow cytometer, sperm sexing