High Sperm Numbers - Detrimental Effects on Bull Sperm During Liquid Storage

Research from the University of Limerick looks at whether high sperm numbers have a detrimental effect on bull sperm during liquid storage.
calendar icon 30 April 2012
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Due to genomic selection, bulls which are in high demand are now entering into artificial insemination (AI) programmes at less than 1 year of age. A typical ejaculate from these bulls will yield 50-150 frozen doses compared to 500-1000 for a mature bull (20 x 106 sperm per dose). To maximise the use of young elite sires during a narrow breeding season it is imperative we develop protocols for the use of liquid semen whereby the sperm number per dose can be reduced without affecting fertility. Currently in Ireland, liquid bull semen contains 5 x 106 sperm per dose, compared to 2 x 106 sperm per dose in New Zealand (Verberckmoes et al, 2005).

In addition, liquid semen has a limited lifespan and is not used at greater than 2.5 days post collection. The hypothesis of this study was that 5 x 106 sperm per dose is counter productive in terms of the generation of oxidative stress, inhibition of sperm mitochondrial activity as well as the consumption of glucose within the diluent and therefore, reducing the sperm number per dose will allow for liquid semen to be stored successfully for longer duration.

Materials and Methods

Semen was collected from Holstein and British Friesian bulls at a commercial AI centre (5 collections with 3-4 bulls per collection; collection = replicate). Sperm concentration was assessed and the ejaculate was diluted in a 5 per cent egg yolk Caprogen diluent (purged in N gas) to a final concentration of 1 (T1), 2 (T2), 3 (T3), 4 (T4) and 5 (T5) x 106 sperm per insemination dose (0.25 mL straws). Semen from each bull was kept separate and each bull was represented in each treatment.

Straws were stored at ambient temperature and assessed in vitro daily for 6 days (Day 0 = day of collection). On each assessment day, semen was evaluated for progressive linear motility (PLM), viability, oxidative stress, mitochondrial activity and glucose consumption. PLM was assessed using standard procedures. Sperm viability, oxidative stress and mitochondrial activity were examined using the fluorescent probes propidium iodide (PI), CM-H2DCFDA and rhodamine 123 (R123), respectively, and analysed using flow cytometry (BDLSR 1; BD Biosciences). Briefly, 1.4 x 106 sperm from each treatment were washed twice in phosphate buffered saline (PBS) at 800g for 10 min and incubated at 37°C in the presence of 125 micrometre CM-H2DCFDA for 30 min followed by 300 micrometre PI for 15 min.

A final wash was performed in PBS to remove excess stain and 10,000 events were analysed. Mitochondrial activity was analysed as above with the exception of a 15 min incubation at 37°C with 2.5 micrometre R123. For glucose analysis samples were centrifuged at 8000g at 4°C and the supernatant was removed, frozen at -20°C and analysed later using a commercial glucose assay kit (Megazyme, Ireland). Briefly, samples were diluted 1:2 with distilled water, added to 1 mL of glucose oxidase/peroxidase reagent and incubated at 45°C for 20 min.

Absorbance was measured on a spectrophotometer and concentrations of glucose were determined using a standard curve. Data were examined for normality, transformed where appropriate and analysed using repeated measures in SPSS (version 20.0). The model included the main effects of day, treatment and day x treatment interactions. Results are presented as mean ± s.e.m.

Results and Discussion

There was no treatment x day interaction for any of the variables assessed. PLM declined with duration of storage from 95.1 ± 0.68 per cent to 73.8 ± 0.99 per cent for all treatments from Day 0 to 5, respectively (P<0.001).

There was an effect of day and treatment on percentage live (P<0.001) with the highest percentage live in T1 and the lowest in T5 on all days (Day 0: 92.4 ± 0.74 and 67.0 ± 2.96 per cent for T1 and T5, respectively, vs. Day 5: 82.8 ± 2.20 and 58.8 ± 3.56 per cent for T1 and T5, respectively. The percentage of live sperm positive for CM-H2DCFDA increased in all treatments on each day of storage (Fig. 1; P<0.001).

There was an effect of treatment (P<0.001) with T1 having the lowest percentage of live sperm positive for CM-H2DCFDA every day and T5 the highest on five days. The percentage of live sperm positive for R123 ranged between 91.6 and 95.8 per cent and was not affected by treatment (P>0.05). The level of glucose in Caprogen declined with duration of storage (P<0.001), being lowest in T5 and highest in T1 on Day 5 (808.6 ± 23.90 and 964.6 ± 18.90 micrograms/mL, respectively).

Fig. 1: The percentage of live bull sperm positive for oxidative stress. Semen was stored at a range of concentration from 1-5 million sperm per dose for 6 days post collection; Vertical bars represent ± s.e.m.


Reducing the sperm number per dose reduces the level of oxidative stress and may be beneficial to the prolonged storage of liquid bull semen.


We gratefully acknowledge support from IRCSET and Enterprise Ireland. Semen was donated by the National Cattle Breeding Centre, Enfield, Co. Meath.


Verberckmoes, S., Van Soom, A., Dewulf, J., de Kruif, A. (2005) Theriogenology 63: 912–922

May 2012
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