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Several laboratories routinely use flow cytometry to evaluate stallion semen quality. However, objective and practical tools for the on-field interpretation of data concerning fertilizing potential are scarce. A panel of nine tests, evaluating a large number of compartments or functions of the spermatozoa: motility, morphology, viability, mitochondrial activity, oxidation level, acrosome integrity, DNA integrity, "organization" of the plasma membrane, and hypoosmotic resistance, was applied to a population of 43 stallions, 33 of which showing widely differing fertilities (19%-84% pregnancy rate per cycle [PRC]). Analyses were performed either within 2 hours after semen collection or after 24-hour storage at 4 °C in INRA96 extender, on three to six ejaculates for each stallion. The aim was to provide data on the distribution of values among said population, showing within-stallion and between-stallion variability, and to determine whether appropriate combinations of tests could evaluate the fertilizing potential of each stallion. Within-stallion repeatability, defined as intrastallion correlation (r = between-stallion variance/total variance) ranged between 0.29 and 0.84 for "conventional" variables (viability, morphology, and motility), and between 0.15 and 0.81 for "cytometric" variables. Those data suggested that analyzing six ejaculates would be adequate to characterize a stallion. For most variables, except those related to DNA integrity and some motility variables, results differed significantly between immediately performed analyses and analyses performed after 24 hours at 4 °C. Two "best-fit" combinations of variables were determined. Factorial discriminant analysis using a first combination of seven variables, including the polarization of mitochondria, acrosome integrity, DNA integrity, and hypoosmotic resistance, permitted exact determination of the fertility group for each stallion: fertile, that is, PRC higher than 55%; intermediate, that is, 45% < PRC less than 55%; or subfertile, that is, PRC less than 45%. Linear regression using another combination of 20 variables, including motility, viability, oxidation level, acrosome integrity, DNA integrity, and hypoosmotic resistance, accounted for 94.2% of the variability regarding fertility and was used to calculate a prediction of the PRC with a mean standard deviation of 3.1. The difference between the observed fertility and the calculated value ranged from -4.2 to 5.0. In conclusion, this study enabled to determine a new protocol for the evaluation of stallion semen, combining microscopical observation, computer-assisted motility analysis and flow cytometry, and providing a high level of fertility prediction., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Spermatozoa from some stallions do not maintain an acceptable fertility after freezing and thawing. The selection of frozen ejaculates that would be suitable for insemination is mainly based on post-thaw motility, but the prediction of fertility remains limited. A recent study in our laboratory has enabled the determination of a new protocol for the evaluation of fresh stallion semen, combining microscopical observation, computer-assisted motility analysis and flow cytometry, and providing a high level of fertility prediction. The purpose of the present experiment was to perform similar investigations on frozen semen. A panel of tests evaluating a large number of compartments or functions of the spermatozoa was applied to a population of 42 stallions, 33 of which showing widely differing fertilities (17–67% pregnancy rate per cycle [PRC]). Variability was evaluated by calculating the coefficient of variation (CV=SD/mean) and the intra-class correlation or “repeatability” for each variable. For paired variables, mean within-stallion CV% was significantly lower than between-stallion CV%, which was significantly lower than total CV%. Within-ejaculate repeatability, determined by analysing 6 straws for each of 10 ejaculates, ranged from 0.60 to 0.97. Within-stallion repeatability, determined by analysing at least 5 ejaculates for each of 38 stallions, ranged from 0.12 to 0.95. Principal component regression using a combination of 25 variables, including motility, morphology, viability, oxidation level, acrosome integrity, DNA integrity and hypoosmotic resistance, accounted for 94.5% of the variability regarding fertility, and was used to calculate a prediction of the PRC with a mean standard deviation of 2.2. The difference between the observed PRC and the calculated value ranged from −3.4 to 4.2. The 90% confidence interval (90CI) for the prediction of the PRC for the stallions of unknown fertility ranged from 8 to 30 (mean = 17). The best-fit model using only motility variables, evaluated after 10 min at 36 °C and 2 h at 36 °C or room temperature, accounted for only 74.2% of the variability. The difference between the observed PRC and the calculated value ranged from −7.2 to 14. The 90CI for the prediction of the PRC for the stallions of unknown fertility ranged from 23 to 48 (mean = 33). In conclusion, this study demonstrated that an appropriate combination of computer-assisted motility analysis, microscopical observation and flow cytometry can provide a higher prediction of fertility than motility analysis alone. [ABSTRACT FROM AUTHOR]

Contents Fertility is reduced after semen cooling for a considerable number of stallions. The main hypotheses include alterations in plasma membrane following cooling and deleterious influence of seminal plasma. However, interindividual variability is controversial. We hypothesized that the removal of seminal plasma could enhance motility in some 'poor cooler' stallions, but could also affect, negatively or positively, membrane quality in some stallions. This study examined the effect of centrifugation, followed or not by removal of seminal plasma, on parameters indicating semen quality after 48 h at 4°C: motility, plasma membrane integrity as evaluated by hypo-osmotic swelling test, acrosome integrity and response to a pharmacological induction of acrosome reaction using ionophore A23187. Sixty-six ejaculates from 14 stallions were used, including stallions showing high or low sperm motility after cooled storage. Centrifugation without removal of seminal plasma did not affect sperm parameters. Removal of seminal plasma did not affect motility, but significantly stabilized sperm membranes, as demonstrated by a higher response to the osmotic challenge, and a reduced reactivity of the acrosome. Moreover, for the same semen sample, the response to an induction of acrosome reaction was significantly higher when the induction was performed in the presence of seminal plasma, compared with the induction in the absence of seminal plasma. This was observed both for fresh and cooled semen. When the induction of acrosome reaction with ionophore A23187 is used to evaluate sperm quality, care must therefore be taken to standardize the proportion of seminal plasma between samples. For the 10 stallions serving at least 25 mares, the only variable significantly correlated with fertility was motility. The influence of membrane stabilization regarding fertility requires further investigations. [ABSTRACT FROM AUTHOR]