Increasing the frequency of ejaculate collection in young dairy bulls increases semen production and field fertility.

Younger bulls typically produce lower volumes of semen per ejaculate with a lower sperm concentration than older more mature, bulls and often fail to meet semen demand using standard collection frequency schedules. The objective of this study was to assess the effect of ejaculate collection frequency on semen output, sperm quality and field fertility in young bulls under commercial conditions. Holstein Friesian bulls aged 366 ± 8 days (mean ± SEM) were assigned one of two ejaculate collection frequencies: (i) HF (n = 14 bulls), where ejaculates were collected twice a day, five days in each two-week period or (ii) LF (n = 12 bulls), where ejaculates were collected once a day, two days per week. The trial period continued until each bull reached both 20 ejaculates and 1000 marketable frozen semen straws. Subjective motility was assessed on all ejaculates pre-freeze and post-thaw (at 0 and 2 h). A subset of ejaculates were assessed post-thaw by computer-assisted sperm analysis for motility, kinematics and morphological defects and by flow cytometry for viability, membrane fluidity, acrosome integrity, reactive oxygen species and DNA fragmentation. A total of 13,846 inseminations (9,541 for HF and 4,305 for LF) were carried out on dairy cows and heifers. HF reached the 1000 straw threshold 41 days earlier than LF (P < 0.01) with the same number of ejaculates. Ejaculate volume and sperm concentration were not affected by treatment but the first ejaculate of the day (HF only) had a greater volume (P < 0.001) and sperm concentration (P < 0.05) than the second ejaculate. HF had higher pre-freeze total (P < 0.01) and gross (P < 0.05) motility than LF. HF had higher post-thaw (2 h) total and gross motility than LF (P < 0.05). Ejaculate rejection rates did not differ between treatments. There was no effect of treatment, week or ejaculate number of the day (HF only) on post-thaw motility and kinematic parameters or sperm viability, membrane fluidity, acrosome integrity and DNA fragmentation. However, HF had lower superoxide production than LF (P < 0.05). Pregnancy per artificial insemination was 64.5 ± 1.0% and 59.9 ± 1.1% for the HF and LF bulls, respectively (mean ± SEM; P = 0.05). In conclusion, collecting ejaculates more frequently from young bulls significantly reduced the number of days required to obtain 1000 straws, increased semen quality in terms of lower superoxide production and increased field fertility.

Effect of storage temperature, nitrogen gassing and sperm concentration on the in vitro semen quality and in vivo fertility of liquid bull semen stored in INRA96.

The aim of this study was to assess the effect of storage temperature, nitrogen (N2) gassing and sperm concentration on in vitro characteristics and calving rate (CR) following artificial insemination (AI) of liquid bull semen stored in INRA96. In Experiment 1 the effect of liquid bull semen diluted in either N2 bubbled or non-bubbled INRA96 at a concentration of 5 × 106 sperm per 0.25 mL insemination dose and stored at 5 or 15 °C was assessed subjectively for total and progressive motility on Days 0, 1, 2, 3 and 4 post collection. In Experiment 2a, the effect of stored liquid semen at three sperm concentrations (3, 4 or 5 × 106 sperm per 0.25 mL insemination dose) on total and progressive motility was assessed subjectively on Days 0, 1 and 2 post collection. In Experiment 2b, the field fertility of liquid semen stored at ambient temperature at a concentration of 3, 4 or 5 × 106 sperm per 0.25 mL dose and inseminated on Days 1 or 2 post collection was assessed in comparison to frozen-thawed semen (total of n = 5742). In Experiment 1, total and progressive motility decreased with increased duration of storage (P < 0.01); however, there was no effect of N2 bubbling on motility on Days 0, 1, 2, 3 and 4 of storage. There was an effect of temperature on total and progressive motility, regardless of treatment, as semen stored at 15°C recorded higher motility values than semen stored at 5°C (P < 0.01). In Experiment 2a, there was no effect of sperm concentration on total or progressive motility on Days 0, 1 or 2 of storage. There was a linear decrease in motility with increased duration of storage (P < 0.01); however, there was no sperm concentration by day interaction. In Experiment 2b, there was an effect of sperm concentration on CR (P < 0.01); semen diluted to 3 and 4 × 106 sperm per dose resulted in a lower CR after 2 days of storage (41.1 and 44.7%, respectively) in comparison to frozen-thawed semen (55.2%) but did not differ to CR of semen diluted to 5 × 106 sperm per dose on Day 2 of storage. There was an effect of parity, fertility sub-index and days in milk (DIM) at AI on CR (P < 0.01). In conclusion, N2 bubbling and sperm concentration had no effect on in vitro sperm motility of liquid semen, but this study demonstrated a reduction in CR on Day 2 of storage at lower sperm concentrations in comparison to frozen-thawed semen.

Effect of increasing equilibration time of diluted bull semen up to 72 h prior to freezing on sperm quality parameters and calving rate following artificial insemination.

An equilibration period of approximately 3-4 h prior to semen cryopreservation is standard practice for maintaining membrane integrity and motility of bull sperm. However, a number of studies indicate that an overnight equilibration period prior to freezing results in improved post-thaw semen quality thus optimising pregnancy rates. The aim of this study was to assess the effect of increasing the equilibration time of bull semen up to 72 h before freezing on sperm quality parameters and calving rate (CR) following artificial insemination (AI) with frozen-thawed semen. The effect of holding semen at 4 °C for 6, 24, 48 or 72 h post dilution before freezing on subsequent post-thaw total and progressive motility (Experiment 1) and field fertility (n = 1640 inseminations, Experiment 2) of frozen-thawed semen was assessed. Equilibration time did not affect post-thaw total and progressive motility (P > 0.05). In addition, there was no effect (P > 0.05) of equilibration time on field fertility with a CR of 53.3, 50.5, 51.3 and 47.3 for the 6, 24, 48 and 72 h treatments, respectively. In conclusion, increasing the equilibration time of diluted bull semen from 6 to 72 h had no significant effect on CR, within the expected range of fertility outcomes, thus providing semen processing centres with flexibility in the time which semen can be held prior to freezing.

Comparison of plant- and egg yolk-based semen diluents on in vitro sperm kinematics and in vivo fertility of frozen-thawed bull semen.

Diluents using components of plant origin have been developed as an alternative to animal based extenders for the dilution of bull semen, however, it is unclear if use of these diluents results in in vivo fertility rates similar to those that occur with use of traditional egg yolk-based diluents. The aim of this study was to assess the effect of semen diluent on 60-day non-return rate (NRR) following artificial insemination (AI) with frozen-thawed bull semen. The effect of semen dilution in one of three different commercial diluents (BullXcell - egg yolk-based, OptiXcell - plant-based or AndroMed - plant-based) on post-thaw total and progressive motility as well as kinematic parameters (Experiment 1) and field fertility (Experiment 2, n = 1,480 inseminations) was assessed. Semen stored in OptiXcell had greater post-thaw total and progressive motility than AndroMed (P < 0.05) but did not differ from BullXcell. Semen stored in BullXcell had a greater beat cross frequency and straight line velocity compared to semen stored in AndroMed (P < 0.05) but did not differ when compared with use of OptiXcell; while values for these variables when using OptiXcell and AndroMed did not differ from each other (P > 0.05). There was no difference in any other sperm kinematic parameters (P > 0.05). There was no effect of diluent on 60-day NRR (71.5%, 67.8% and 70.6% for BullXcell, OptiXcell and AndroMed, respectively). In conclusion, while diluent significantly affected post-thaw sperm motility and kinematics, no effect on 60-day NRR was observed. Given that OptiXcell and AndroMed are animal protein-free media these diluents may be a suitable alternative to BullXcell for the storage of frozen-thawed bull semen.