Small ruminant SexedULTRA™ sperm sex-sorting: Status report and recent developments.

Flow cytometry sperm sex-sorting based on the relative DNA difference between X- and Y-chromosome bearing populations is an established method that has allowed the production of pre-sexed offspring in a multitude of species and has been a commercial success in cattle production for the past twenty years. Several improvements to the technology and the processing methods have increased the sorting efficiency of ejaculates and the post-thaw quality of sex-sorted sperm, allowing for the fertility gap between conventional (non-sorted) and SexedULTRA™ sex-sorted sperm to be bridged. Small ruminant industries are now progressively testing sex-sorted sperm for application in their specific niches and environments. A review of the key advances and the recent developments in caprine, ovine and cervine sperm sex-sorting technology are described in this publication.

In vitro sperm quality and DNA integrity of SexedULTRA™ sex-sorted sperm compared to non-sorted bovine sperm.

SexedULTRA™ is an improved method of sex-sorting sperm creating a less damaging environment to retain sperm integrity through the sorting process. The aim of this study was to evaluate the in vitro characteristics of fresh and frozen bovine sperm using the SexedULTRA™ method, and compare it to conventional (non-sorted) sperm. For both methods, percent total sperm motility was estimated visually and also classified into total and progressively motile using a computer assisted sperm analyzer (CASA). Percent sperm with intact plasma membranes (VIA) and acrosomes (PIA) were assessed using flow cytometry and sperm DNA fragmentation index (DFI) was estimated using the Bull sperm Halomax® Kit. Two contemporaneous ejaculates from 10 bulls were processed and cryopreserved using one of the two procedures (SexedULTRA™ and conventional). Sperm motility, VIA and PIA were assessed post-thaw (0 h) and post-incubation (3 h at 37 °C, 8 h and 24 h at 18 °C). DFI was analyzed post-thaw (0 h) and after 6, 24, 48 and 72 h of incubation at 37 °C. In a second experiment, ejaculates from 7 bulls were split sampled into the two types of processing (SexedULTRA™ and conventional) and diluted using a fresh semen extender developed for sex-sorted bovine sperm. Sperm quality was assessed after dilution (0 h) and after incubation for 12, 24, 48, 72 h at 18°, and the same time points of incubation at 37 °C for DFI. Frozen-thawed SexedULTRA™ sperm was significantly (P < 0.05) better than conventional semen after a 3 h incubation at 37 °C for PIA, and after a 24 h incubation at 18 °C for percent visual motility and PIA. DFI was significantly lower for SexedULTRA™ compared to conventional at all time points of incubation (37 °C). Fresh SexedULTRA™ sperm showed improved quality compared to conventional at all time points of incubation at 18 °C for percent visual and total motile sperm, VIA, PIA, and DFI. Significant differences were also found in progressive motile sperm immediately after dilution (0 h), but not at any time point after incubation. The results show that the SexedULTRA™ process maintains the quality of sex-sorted sperm and, in many cases, has better in vitro longevity than conventional semen.

Is sperm DNA fragmentation a good marker for field AI bull fertility?

This paper aimed at investigating the potential use of sperm DNA fragmentation (SDF) to improve the routine screening of infertility of Holstein bulls. Cryopreserved sperm samples from 201 Holstein bulls provided by an AI center were used in the analyses of SDF at 0 (SDF_0) and 6 (SDF_6) h of incubation at 37°C. A refinement of the sperm chromatin dispersion test implemented in the Sperm-Halomax kit was employed to measure SDF. Records on routinely collected semen traits (volume, concentration, mass and individual motility evaluated in the fresh ejaculate, and individual motility in post-thawed semen straws) were provided by the AI center. Artificial insemination bull fertility was obtained from official field recording as successful or failed insemination. The results show that the average SDF was low (around 3.5%) at 0 and 6 h of incubation. A moderate effect of inbreeding depression was found. Estimated heritability for SDF traits were moderately high (0.41 and 0.29 for SDF_0 and SDF_6, respectively) and estimated repeatability of SDF measures in the same animal were high (0.73 and 0.70 for SDF_0 and SDF_6, respectively). An overall estimated service bull value (ESBV) obtained through statistical modeling that allowed for adjustment of systematic environmental effects not specific to a bull and of the female contribution to fertility, and the estimated genetic values (EGV) were obtained from field-recorded AI information. The ESBV and EGV were also obtained for all semen traits. Moderately large and negative Pearson correlation coefficients were observed between SDF traits and male fertility ranging from (-0.43 to -0.50; P <0.001). Results of stepwise regression analyses showed that SDF_6 had the largest partial r(2) (0.15 to 0.26) among all semen characteristics. Overall, the selected semen traits explained 25% and 31% of the observed variability in bull fertility measured as EGV and ESBV, respectively. When looking at the predictive ability of bull fertility categories, the results of discriminant and logistic regression analyses showed that low-fertility bulls (those in the 10th or lower percentile in the fertility distribution) can be accurately identified by using measures of SDF alone or in combination with sperm motility. Values of SDF around 7% to 10% could be used as indicators of low AI success.

DNA fragmentation kinetics and postthaw motility of flow cytometric-sorted white-tailed deer sperm.

This study examined DNA damage and postthaw motility of white-tailed deer sperm (n = 28) before and after sex selection and conventional sorting using MoFlo XDP SX flow cytometry. Semen samples from the same individuals were treated in 4 different ways: 1) chilled-extended sperm samples (without glycerol); 2) cryopreserved conventional samples, samples directly cryopreserved after the addition of extenders; 3) cryopreserved conventionally sorted samples, sorted samples to remove the dead sperm subpopulation; and 4) cryopreserved sex-sorted samples; sorted samples to remove the dead sperm subpopulation and separation of X- and Y-chromosome-bearing sperm. In all the cases (n = 6), conventional samples showed decreased postthaw motilities (43 ± 26%) when compared with X-sorted samples (59 ± 20%; P < 0.05) and Y-sorted samples (54 ± 20%; P > 0.05). The DNA fragmentation baseline was <5% for frozen-thawed conventional samples, but even less after sex sorting and conventional sorting: 2.4 and 1.7%, respectively. On the other hand, conventional samples showed greater (P < 0.05) DNA fragmentation than the sex-sorted sperm (n = 6) at 96 h (average of 4.8 ± 4.5% and 5.3 ± 4%, respectively). Conventionally sorted samples (n = 8) did not have greater (P > 0.05) DNA fragmentation when compared with the sex-sorted samples. Fragmentation of DNA on X-chromosome and Y-chromosome-bearing sorted sperm were not significantly different (n = 10, P > 0.05) after 96 h (2.6 ± 3.6% and 2.2 ± 0.5%, respectively). Future research should be implemented for examining the fertilizing potential of sex-sorted white-tailed deer sperm (e.g., AI fertility trials).

Bacteria in bovine semen can increase sperm DNA fragmentation rates: a kinetic experimental approach.

Cryopreserved straws of semen (n=228) from Holstein bulls (n=47) were examined for bacterial presence and sperm DNA fragmentation (SDF) dynamics. Commercial semen doses (representing six ejaculates per individual) were randomly selected from a bull stud in Spain. The dynamics of SDF were assessed after thawing (T0) and at 4, 24, 48, 72 and 96h of incubation at 37°C, using the commercial variant of the sperm chromatin dispersion test for Bovine (Halomax®). One group of bulls showed a bacterial presence in semen samples between 0 and 96h of incubation (n=23, group A) while the other did not (n=24, group B). Immediate post-thaw differences in SDF were not observed when both groups were compared. However, the rate of increase in SDF (rSDF) over time, considered as an estimate of the kinetic behaviour of sperm DNA survival, was significantly higher (P<0.05) in semen samples from group A (0.7% per hour) versus group B (0.05% per hour). Polymerase Chain Reaction (PCR) assay was used for DNA amplification using primers designed for specific regions of the bacterial gene that codifies for 16S rRNA. Different species within the phyla Bacteroidetes, Firmicutes, Proteobacteria, Cyanobacteria, Fusobacteria and Actinobacteria were identified. The results show that (1) SDF at baseline (T0) may not be affected by the presence of bacteria but the rSDF can increase due to bacterial growth during incubation, (2) the increase in the rSDF is characteristic of some bulls but not for others, and (3) certain bacterial strains are repeatedly found in separate ejaculates from the same bull.