330 THE EFFECT OF SHEATH FLUID ON THE QUALITY OF SEX-SORTED RAM SPERMATOZOA
S.P. de Graaf A , L. Gillan A , G. Evans A , W.M.C. Maxwell A and J.K. O’Brien AReproGen, Faculty of Veterinary Science, The University of Sydney, NSW 2006, Australia. email: simong@vetsci.usyd.edu.au
Reproduction, Fertility and Development 16(2) 284-285 https://doi.org/10.1071/RDv16n1Ab330
Submitted: 1 August 2003 Accepted: 1 October 2003 Published: 2 January 2004
Abstract
The fertility of sexed frozen-thawed ram spermatozoa at low AI doses remains below commercially acceptable levels (Maxwell WMC et al. 2003 Theriogenology 59, 511 abst). The aim of the present study was to determine if the use of an artificial seminal plasma sheath fluid could attenuate the stress of dilution during flow cytometry and thus improve the functional capacity of sexed ram spermatozoa. Semen was collected from 3 Merino rams, processed for sex-sorting (Hollinshead FK et al. 2002 Reprod. Fertil. Dev. 14, 503–508), and then allocated to 2 treatment groups according to the sheath fluid used within the flow cytometer: (i) a tris-citrate-fructose diluent (TRIS), or (ii) an artificial seminal plasma diluent (ASP) (ODonnell JM 1969 J. Reprod. Fert. 19, 207–209). Spermatozoa were sorted using a modified high speed cell sorter (SX MoFlo®, DakoCytomation, Fort Collins, USA) without separation of X and Y bearing gametes, and then frozen and thawed using established protocols (Hollinshead FK et al. 2002). Motility characteristics (HTM-IVOS; Hamilton-Thorne, Beverly, USA) and acrosome integrity (FITC-PNA) were assessed throughout. Statistical analyses were conducted by ANOVA. Results indicate that in all but one instance (VSL pre-freeze), TRIS spermatozoa exhibited higher (P < 0.05) total motility (TM), average path velocity (VAP), straight line velocity (VSL), curvilinear velocity (VCL) and amplitude of lateral head displacement (ALH) compared to ASP spermatozoa (Table 1). Conversely, ASP treatments displayed enhanced straightness (STR) and linearity (LIN) pre-freezing and immediately post-thaw. In addition, the percentage of hyperactivated (HA) spermatozoa prior to freezing (± SEM) was lower (P < 0.05) for ASP (3 ± 1.1) than for TRIS (20 ± 3.4), although post-thaw the differences were NS. Acrosome integrity remained similar (P > 0.05) among treatments before and after thawing (range: 92.3–95.9% intact). In conclusion, the ASP sheath fluid resulted in decreased TM, velocity and ALH of sex-sorted frozen-thawed ram spermatozoa, a clear indication of the superiority of TRIS sheath fluid. The partial improvement in STR, LIN and HA that the ASP affords sex-sorted ram spermatozoa does not offset the aforementioned negatives, suggesting ASP is unsatisfactory for use as a sheath fluid during sperm sorting. Research supported by XY, Inc.
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