Comparison of sperm adenosine triphosphate content, motility and fertility of immobilized and conventionally cryopreserved Norwegian Red bull semen.

Estrus detection and timing of AI remains a challenge in cattle breeding. Prolonging spermatozoa lifespan after AI, making sperm cells available over an extended period, could make timing of AI relative to ovulation less crucial and improve fertility. Immobilization of sperm cells by the patented SpermVital technology in an alginate gel will provide a gradual release of spermatozoa after AI. The first aim of this study was to examine fertility, measured as non-return rate after 56 days (NR56), of SpermVital (SV) processed semen with reduced sperm cell number per dose compared to earlier studies, and compare with conventionally processed semen in Biladyl, a proprietary version of the egg yolk Tris semen extender. The second aim was to examine in vitro sperm quality post-thaw and after thermal stress. The third aim was to examine potential correlations between in vitro sperm parameters and NR56. Ejaculates from 16 Norwegian Red young bulls were split in three, processed and cryopreserved as Biladyl semen (B15; 15 million spermatozoa/dose) or by SpermVital technology (SV25; 25 million spermatozoa/dose or SV15; 15 million spermatozoa/dose). 1400 semen doses were produced per bull and distributed throughout Norway for a blinded field trial. Fertility was recorded as NR56 after first AI (N = 7155). Two ejaculates from each bull were randomly selected for in vitro experiments. B15 and SV15 semen samples were analyzed for motility by computer-assisted sperm analysis, viability and acrosome integrity by flow cytometry and ATP content by bioluminescence assay, post-thaw and after thermal stress. The AI trial detected no differences in NR56; least square means being 75.5% (B15), 75.6% (SV25) and 74.8% (SV15) (p > 0.05). There were no differences in total motility and progressive motility post-thaw, however, after three hours incubation at 38 °C, SV sperm motility and progressivity were higher for SV15 than for B15 spermatozoa (p < 0.05). The percentage of acrosome intact live sperm cells was higher for SV15 than B15 spermatozoa at all timepoints analyzed (0 h, 3 h, 24 h, p < 0.05). B15 semen showed a higher ATP level than SV15 at 0 h (p < 0.05), while SV15 sperm cells had higher ATP levels after 3 and 24 h (p < 0.05). No association was detected between in vitro sperm parameters and NR56. In conclusion, SV15, SV25 and B15 semen yielded equal fertility after AI. However, there were differences in sperm quality, as SV15 spermatozoa displayed higher motility, viability and ATP levels after thermal stress than B15 spermatozoa (p < 0.05).

Use of immobilized cryopreserved bovine semen in a blind artificial insemination trial.

To make timing of artificial insemination (AI) relative to ovulation less critical, methods for prolonging shelf life of spermatozoa in vivo after AI have been attempted to be developed. Encapsulation of sperm cells is a documented technology, and recently, a technology in which sperm cells are embedded in alginate gel has been introduced and commercialized. In this study, standard processed semen with the Biladyl extender (control) was compared with semen processed by sperm immobilization technology developed by SpermVital AS in a blind field trial. Moreover, in vitro acrosome and plasma membrane integrity was assessed and compared with AI fertility data for possible correlation. Semen from 16 Norwegian Red young bulls with unknown fertility was collected and processed after splitting the semen in two aliquots. These aliquots were processed with the standard Biladyl extender or the SpermVital extender to a final number of 12 × 10(6) and 25 × 10(6) spermatozoa/dose, respectively. In total, 2000 semen doses were produced from each bull, divided equally by treatment. Artificial insemination doses were set up to design a blinded AI regime; 5 + 5 straws from each extender within ejaculates in ten-straw goblets were distributed to AI technicians and veterinarians all over Norway. Outcomes of the inseminations were measured as 56-day nonreturn rate (NRR). Postthaw sperm quality was assessed by flow cytometry using propidium iodide and Alexa 488-conjugated peanut agglutinin to assess the proportion of plasma membrane and acrosome-intact sperm cells, respectively. In total, data from 14,125 first inseminations performed over a 12-month period, 7081 with Biladyl and 7044 with SpermVital semen, were used in the statistical analyses. There was no significant difference in 56-day NRR for the two semen categories, overall NRR being 72.5% and 72.7% for Biladyl and SpermVital, respectively. The flow cytometric results revealed a significant higher level of acrosome-intact live spermatozoa in Biladyl-processed semen compared to SpermVital semen. The results indicate that the level of acrosome-intact live spermatozoa in the AI dose did not affect the 56-day NRR for the two semen processing methods. In conclusion, this study has showed that immobilized spermatozoa provide equal fertility results as standard processed semen when AI is performed in a blinded field trial, although the immobilization procedure caused increased sperm damage evaluated in vitro compared to standard semen processing procedure.

Comparison of electronic volume and forward scatter principles of cell selection using flow cytometry for the evaluation of acrosome and plasma membrane integrity of bull spermatozoa.

The objective of the study was to compare two different flow cytometers to reveal if there are differences between them and to find the most suitable protocol for analysis of spermatozoa. These two flow cytometers; Cell Lab Quanta™ and Coulter Epics XL, have different principles to calculate cell size, electric volume, and forward scatter (FS), respectively. Flow cytometry is a valuable tool to assess various spermatozoa quality traits simultaneously, such as plasma membrane and acrosome integrity. A double- and triple-stain combination was performed to compare evaluation of these two parameters by both flow cytometers and to assess the need of a fluorescent probe to identify the spermatozoa. Propidium iodide was used to assess the proportion of dead spermatozoa, whereas Alexa Fluor(®) 488 conjugated peanut agglutinin (PNA- Alexa 488) was used to evaluate the percentage of acrosome intact and acrosome-reacted cells or degenerated cells. In the triple-stain protocol, MitoTracker(®) Orange (MO) was included to test the capacity of this probe to discriminate spermatozoa from egg yolk and debris particles present in the semen sample. Cryopreserved semen from 13 Norwegian Red bulls was included in the study and the semen was evaluated immediately after thawing and after 3 hr incubation at 37°C. The results show that there is good agreement between the instruments. Nevertheless, a significant difference was found in percentages of acrosome intact live spermatozoa (% AIL) when including MO as a spermatozoa identification probe, compared to assessment without MO, with the Coulter Epics XL, while no significant difference was found when including the probe with the Cell Lab Quanta. In conclusion, the results show that cell size measurement based on electronic volume used by the Cell Lab Quanta flow cytometer is more accurate than FS used by the Coulter Epics XL flow cytometer in identification of spermatozoa.