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CHAPTER 4.1
centrifugation (T1) and 72 h (T4) after cooled storage, an aliquot of each tube was examined for DNA
integrity using TUNEL assay.
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4.1.3.7. Statistical analysis
EXPERIMENT 1: influence of centrifugation protocol on function of fresh and cooled sperm
1/ sperm loss
To evaluate whether the treatment (CP 1, 2, 3, 4 and 5) had an influence on sperm
concentration in the aspirated supernatant, a Kruskal Wallis H test was conducted (overall treatment
effect) as well as a Mann-Whitney U test (to test the difference between CP 1 versus the other
treatments, separately) using SPSS software package (version 17.0, SPSS Inc., Chicago, IL).
2/ effect of centrifugation (non CP vs CP)
Mixed models were fitted in SAS 9.1.3. (PROC MIXED) with centrifugation (non CP vs CP),
time (T1- T4) and interaction centrifugation × time included as predictor variables. Stallion (1-5) and
ejaculate (5 per stallion) were included as random effects to account for clustering of ejaculate in
stallion and the repeated measurements over time, within ejaculate (including an AR(1) correlation
structure), respectively. The outcome variables (percentage membrane intact sperm cells,
percentage acrosome intact sperm cells, BCF, TM and PM) were normally distributed based on the
inspection of QQ-plots and Kolmogorov-Smirnov tests.
3/ effect of centrifugation protocol
Mixed models were fitted in SAS 9.1.3. (PROC MIXED) with CP (1-5), time (T1- T4) and the
interaction CP × time included as predictor variables. Stallion (1-5) and ejaculate (5 per stallion) were
included as random effects to account for clustering of ejaculate in stallion and the repeated
measurements over time, within ejaculate (including an AR(1) correlation structure), respectively.
The outcome variables (percentage membrane intact sperm cells, percentage acrosome intact sperm
cells, BCF, TM and PM) were normally distributed based on the inspection of QQ-plots and
Kolmogorov-Smirnov tests.