|Abstract:||Artificial insemination is a common practice within the swine industry and accurate semen evaluation is necessary to maintain reproductive efficiency. Additionally, identifying the cause of reduced fertility in sex-sorted sperm could enhance the industry. Our previous work has shown that sperm binding to oviduct cell aggregates improves fertility prediction, possibly by estimating the ability of sperm to form a reservoir in the oviduct. We have also shown that two oviduct glycan motifs, biantennary 6-sialylated N-acetyllactosamine (bi-SiaLN) and LewisX trisaccharide (LeX) bind boar spermatozoa with high affinity and specificity. Measuring binding to soluble glycans is less laborious than assessing binding to oviduct cell aggregates and more suitable for industry use. The objective of the first study was to determine if the ability to bind oviduct cells or the specific oviduct glycans that sperm bind are useful supplements to routine semen analyses. A fertility trial using semen from boars (n=30) used for homospermic matings was designed. Semen was collected and shipped overnight for laboratory analysis and for inseminations. Semen collections were analyzed from boars three times over the course of the study. Oviduct cell binding, motility, morphology and acrosome status were assessed the day the shipment was received. Binding to three soluble fluoresceinated glycans bi-SiaLN, sulfated LeX (suLeX), and the control lactosamine disaccharide (LacNAc) was measured the following day. The inseminations occurred at 15 farms in the greater Midwest and farrowing data from all matings from each boar were used (>50 matings/boar). Pregnancy rate (PR) and litter size (LS) were adjusted to account for different farms, number of services, number of doses inseminated, and sow parity, using the MIXED procedure in SAS 9.4. A fertility index (FI) was generated, consisting of PR x LS. This index allowed for boars to be ranked on combined PR and LS. Lastly, the GLMSELECT procedure was used to select variables having a significant impact on PR, LS, and FI. The predictive models constructed were further analyzed using the REG procedure and accounted for 58% or more of the variation in PR, LS, and FI [PR (p < 0.001, r2 = 0.60), LS (p < 0.001, r2 = 0.58), and FI (p < 0.001, r2 = 0.63)]. All three final models included traditional semen morphology evaluation traits. The model for PR also included the ability of sperm to bind oviduct cell aggregates, in agreement with our previous smaller scale study. However, binding to specific oviduct glycans was not a useful supplement to traditional semen analysis. The objective of the second study was to determine how sorting sperm by flow cytometry to separate X and Y chromosome-bearing sperm affects binding to oviduct cells and purified oviduct glycans. The sperm rich fraction from boars (n=5) was collected; sperm were stained with Hoechst 33342 and sorted. Sperm were separated into either X or Y chromosome-bearing cells and placed into the following treatments: 1) sperm sorted for the X chromosome, 2) sorted for the Y, 3) an equal mixture of sorted X and Y, and 4) a control of non-sorted sperm from the same collection. Samples were delivered and tested for oviduct cell binding within 12 hr of sorting. Additionally, we observed motility characteristics, acrosome status, and glycan binding to the three soluble fluoresceinated glycans previously mentioned. Results showed that the number of sperm binding to oviduct cells was reduced by more than half in the three sorted samples compared to the control. There were no differences observed between samples or individual glycans when binding of fluoresceinated soluble glycans was investigated. The percentage of sperm that were motile in the sorted samples was reduced on average by 15% from the unsorted control. All samples maintained >97% acrosome integrity after the sorting process. In conclusion, sperm binding to the complex matrix around oviductal cell aggregates was reduced after sorting but binding to purified soluble fluoresceinated glycans was not different among sperm preparations. Overall, binding to oviduct cell aggregates was correlated with pregnancy rate and can be used in conjunction with other traditional sperm assays to predict the average pregnancy rate of an individual boar. Similarly, a reduction in fertility in sperm that are sex-sorted may be due to a reduced ability to bind to the oviduct epithelium. These results show that the interaction with oviductal epithelial cells remains impactful as a measure for potential sperm success within the female reproductive tract.