14 Sperm membrane integrity in ejaculates from young bulls may be improved by single-layer centrifugation

Abstract

Sperm quality in the first ejaculates of young bulls is often poor, leading to rejection of these samples for commercial freezing. It may be several weeks before sperm quality reaches the thresholds regarded as being acceptable for commercial production. Single-layer centrifugation (SLC) has been shown to separate robust bull spermatozoa from the rest of the ejaculate (Goodla et al. 2015 J. Dairy Sci. 97, 2204-2212; Nongbua et al. 2017 Reprod Domest. Anim. 52, 596-602). The aim of this study was to test whether SLC can be used to select spermatozoa from the ejaculates of young bulls and thus freeze samples earlier than is currently possible. Ejaculates (at least 3 per bull; n=33) were collected from 9 young Holstein or Swedish Red bulls, 255 to 415 days old. The sperm concentration was adjusted to 69×106 spermatozoa mL−1 with Andromed™ (Minitub, Tiefenbach, Germany) before layering part of the sample over 4mL of Bovicoll colloid. The remaining sample served as the unselected control. After centrifugation at 300×g for 20min, the sperm pellet was aspirated into fresh extender. Both control (uncentrifuged) and SLC samples were frozen and stored in LN pending analysis. Membrane integrity was evaluated by flow cytometry after staining for 10min at 37°C with SYBR14/propidium iodide (Live-Dead Sperm Viability Kit, L-7011; Invitrogen, Carlsbad, CA, USA). Paired t-test was used to compare results for control and SLC samples, and Pearson correlation was used for age and membrane integrity. The median age at which the samples were taken was 319 days (range: 255-415 days). Membrane integrity varied considerably among bulls (range: 11-72%). The mean proportion of membrane-intact spermatozoa in controls and SLC samples was 40±15 and 47±16%, respectively (P<0.01). In 21 of the 33 ejaculates (64%), the SLC sample had a higher membrane integrity than the controls (Table 1). Age in days was positively correlated with membrane integrity for SLC samples (r=0.40; P<0.05) but not for controls. These results suggest that SLC might be a useful technique for selecting membrane-intact spermatozoa from the ejaculates of young bulls. However, there is considerable variation among bulls, possibly due to age, which is currently being investigated. Other parameters of sperm quality, such as chromatin integrity, mitochondrial membrane potential reactive oxygen species production, and motility, are also being evaluated. Table 1.Age and membrane integrity (mean±standard deviation) in ejaculates from young bulls before and after SLC (n=33) This project was funded by the Royal Swedish Academy of Agriculture and Forestry, Viking Genetics, and the Faculty for Veterinary Medicine and Animal Health, SLU. We thank Viking Genetics for supplying the semen samples.