Current in vitro tests of sperm function are not highly correlated with male fertility, and homospermic inseminations require hundreds of inseminations per treatment to obtain accurate fertility data. Heterospermic insemination, mixing the sperm of two or more males, provides an accurate estimation of relative fertility in most species examined. In this study, a heterospermic method was developed to test in vivo fertility of bulls rapidly. Frozen, flow-sorted sperm from 4 groups of 4 bulls were thawed. Sperm from 3 bulls within each group were combined in all possible combinations (ABC, ABD, ACD, BCD) and inseminated into heifers 12 or 24 h following onset of estrus. Equal numbers of progressively motile sperm were inseminated from each bull, totaling 600,000 motile sperm post-thaw. Half of each inseminate was deposited into each uterine horn. Embryos were collected nonsurgically 14.5 to 20 days following estrus. Collections yielded 165 elongating embryos from 332 heifers (48%) with no difference between AI 12 or 24 h post estrus. Polymorphic DNA markers were used to genotype embryos to determine the sire of each embryo biopsy. After genotyping, 118 of the 165 embryos could be assigned a specific sire. Heterospermic indices for ranking each bull within groups were calculated using the maximum likelihood analysis theorem (Table 1). In group 1, the fertility of the poorest bull was significantly lower (P<0.05) than two other bulls. In group 2, the dominant bull had the highest index, and therefore the highest fertility in the group (P<0.05). Similar distinctions could be made in groups 3 and 4. However, in three of the groups the fertility of some bulls was not clearly high or low (P>0.05).
Table 1. Heterospermic indices + SE for individual bulls within groups.
| Group 1 | Group 2 | Group 3 | Group 4 |
|---|---|---|---|
| 1.47 + .41a | 2.43 + .43a | 1.68 + .44a | 0.92 + .36a,b |
| 0.44 + .27a,b | 0.22 + .15b | 1.09 + .39a,b | 0.46 + .20a |
| 1.84 + .46a | 0.90 + .35b | 0.83 + .31a,b | 2.02 + .40b |
| 0.25 + .17b | 0.45 + .23b | 0.40 + .22b | 0.59 + .24a |
With these procedures, an average of 30 genotyped embryos per group of 4 bulls enabled detection of bulls with clearly differing fertility. This in vivo test requiring few females rapidly provides information concerning which bulls have relatively high or low fertility. In addition, sperm treatments could be evaluated with this technique.
Acknowledgment: This research was supported by XY, Inc., Fort Collins, CO,
USA.