Elsevier

Theriogenology

Volume 77, Issue 8, May 2012, Pages 1597-1604
Theriogenology

Research article
Sperm viability, motility and morphology in emus (Dromaius novaehollandiae) are independent of the ambient collection temperature but are influenced by storage temperature

https://doi.org/10.1016/j.theriogenology.2011.11.028Get rights and content

Abstract

A protocol for storage of emu semen >6 h has not yet been optimized. The objective was to determine: a) whether sperm quality was adversely affected by sudden exposure to low temperatures (5, 10 and 20 °C) during collection; and b) the effects of three storage temperatures (5, 10 and 20 °C) on survival of emu sperm. In two experiments, each repeated three times on alternate days, ejaculates were diluted 1:1 with precooled (5, 10, or 20°C) UWA–E3 diluent and stored for up to 48 h. Collection temperature, or interaction with either the storage time or storage temperature, had no significant effect on sperm viability, motility, or morphology. Mass Motility Score (2.91–3.27 ± 0.26, mean ± SEM), and percentages of live (72.4–76.2 ± 2.4) and morphologically normal sperm (63.3–64.5 ± 2.3) were comparable among collection temperatures. Conversely, storage temperature and storage time affected (P < 0.05) sperm viability, motility, and morphology. After storage for 48 h, percentages of viable, normal, and motile sperm were higher (P < 0.001) at 5 °C (58.7% ± 1.1, 44.7% ± 1.3, and 50.7% ± 4.9, respectively) and 10 °C (62.6% ± 1.1, 54.1% ± 1.3, and 60.4% ± 4.9) than at 20 °C (27.6% ± 1.1, 20.1% ± 1.3, and 25.9% ± 4.9). Beyond 6 h of storage, the percentage of abnormal sperm was higher (P < 0.001) for storage at 5 °C compared to 10 and 20 °C. After 48 h, bacterial counts were considerably higher at 20 °C compared to 5 and 10 °C (P < 0.001). The pH of stored sperm suspension remained unaffected at 5 and 10 °C, but at 20 °C declined to 6.5 ± 0.03 after 24 h (P < 0.05) and to 6.0 ± 0.03 after 48 h (P < 0.001). We concluded that emu semen could be collected at low ambient temperatures (5–20 °C) without compromising its in vitro storage duration and that semen quality during storage for 48 h was better if it was stored at 10 °C than at 5 or 20 °C.

Introduction

Exposing sperm to subphysiological temperatures, especially in the range of 0 to 20 °C may cause damage, or ‘chilling injury’ [1]. This may occur immediately upon cooling (direct chilling injury), or as an indirect chilling injury inflicted over an extended storage interval [2]. Sperm may be injured at the time of semen collection and during hypothermic in vitro storage and may be susceptible to the thermotrophic phase transition of the sperm plasma membrane [3]. Accordingly, adequate precautions are necessary to ensure that sperm are handled to minimize chilling injury. Hence, for bulls and boars, semen is usually collected in insulated containers [4], [5] and stored at temperatures higher than those which might induce cold-shock and death—for example, >12 °C for boar semen[6]. For the chicken and turkey, failure to comply with these conditions may induce irreversible loss of sperm viability, as evidenced by the 10% decline in fertility when semen is collected into dry tubes at 5 °C compared to 15 and 25 °C [7], [8]. For the bull, ram, rabbit, dog, boar and stallion, there are irreversible declines in motility, sperm survival and fertilizing capacity as semen is rapidly cooled to < 15 °C before freezing [9], [10], [11], [12].

The issue of collection temperature could be especially relevant for emu (Dromaius novaehollandiae), since its physiological breeding season is in winter [13]. To date, semen has been collected directly into vials that are exposed to ambient temperatures that are often 5 to 15 °C. Considerable loss of sperm membrane integrity and fertilizing ability occurred after cooling and storage of undiluted emu semen at 4 °C [14]. Further, sperm viability, motility, membrane integrity, and fertilizing ability were all reduced after 6 h storage at 4 and 20 °C following collection at ambient temperature and dilution with Lake's and BPSE poultry semen diluents [14]. Even using ‘Emu’ diluent, storage could not sustain sperm viability beyond 24 h [14]. By contrast, the quality of chicken or turkey semen can be maintained for 24 to 48 h [15], [16], [17].

That emu sperm function cannot be sustained beyond 6 h suggests susceptibility of emu sperm to chilling injury when suddenly exposed to subphysiological temperatures, either ambient temperatures at ejaculation, or when it is stored at 4 °C. To determine whether the low temperatures at which semen is either collected or stored cause chilling injury, we tested the hypothesis that low collection temperature would decrease sperm function during liquid storage while the storage temperature will inversely affect sperm function.

Section snippets

Materials and methods

Two experiments were conducted in the second half of the emu breeding season (late July to early August), following approval by the Animal Ethics Committee (Approval No: RA/3/100/775). In Experiment 1, semen was collected at 5, 10, or 20 °C, simulating the typical range of ambient temperatures during semen collection. For individual males, the effect of collection temperature on sperm viability, motility and morphology during 48 h storage at 5, 10 and 20 °C were investigated. In Experiment 2,

Experiment 1

Collection temperature did not significantly affect sperm functions (motility and viability) or sperm morphology. The means amongst different collection temperatures were not significantly different: 3.3 ± 0.20 for the Mass Motility Score, 74.9 ± 1.3 for percentage of total live, 63.7 ± 0.4 for percentage of live normal, and 11.2 ± 1.1 for percentage of live abnormal sperm.

In addition, there were no significant interactions of collection temperature with either storage time or storage

Discussion

The findings of this study did not support the hypothesis that a low temperature for the collection vessel at the time of semen collection would reduce sperm quality. However, with respect to subsequent storage, sperm quality was strongly influenced by the temperature and duration of storage, with adverse outcomes particularly evident after 6 h at 20 °C.

The tolerance of emu sperm to low collection temperatures may be attributed to the high percentage of unsaturated fatty acids (heneicosanoic,

Acknowledgments

We thank Jason Ledger for technical assistance in the laboratory and the staff of the Animal Care Unit of the University of Western Australia for their cooperation. The Rural Industries Research and Development Corporation (Australia) supported this study. We are also thankful to the Endeavor Awards, administered by the Department of Education, Employment and Workplace Relations government of Australia, for providing the post graduate scholarship to the corresponding author that enabled him to

References (45)

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