Cluster analysis reveals a binary effect of storage on boar sperm motility function

Abstract

Storage of liquid-preserved boar spermatozoa is associated with a loss of fertilising ability of the preserved spermatozoa, which standard semen parameters barely reflect. Monitoring responses to molecular effectors of sperm function (e.g. bicarbonate) has proven to be a more sensitive approach to investigating storage effects. Bicarbonate not only initiates capacitation in spermatozoa, but also induces motility activation. This occurs at ejaculation, but also happens throughout passage through the oviduct. In the present study we tested whether the specific response of boar sperm subpopulations to bicarbonate, as assessed by motility activation, is altered with the duration of storage in vitro. Three ejaculates from each of seven boars were diluted in Beltsville thawing solution and stored at 17°C. Only minor changes in the parameters of diluted semen were revealed over a period of 72h storage. For assessment of bicarbonate responses, subsamples of diluted spermatozoa were centrifuged through a discontinuous Percoll gradient after 12, 24 and 72h storage. Subsequently, spermatozoa were incubated in two Ca2+-free variants of Tyrode's medium either without (TyrControl) or with (TyrBic) 15mM bicarbonate, and computer-aided sperm analysis motility measurements were made. Cluster analysis of imaging data from motile spermatozoa revealed the presence of five major sperm subpopulations with distinct motility characteristics, differing between TyrBic and TyrControl at any given time (P<0.001). Although there was an increasing loss of motility function in both media, bicarbonate induced an increase in a 'fast linear' cohort of spermatozoa in TyrBic regardless of storage (66.4% at 12h and 63.9% at 72h). These results imply a binary pattern in response of sperm motility function descriptors to storage: although the quantitative descriptor (percentage of motile spermatozoa) declines in washed semen samples, the qualitative descriptor (percentage of spermatozoa stimulated into fast linear motion by bicarbonate) is sustained independent of the duration of storage.

Dinámicas espermáticas subpoblacionales durante el proceso de criopreservación en eyaculados de caprino (Capra aegagrus hircus)

“…En el presente estudio, se han analizado los efectos de las diferentes etapas de manipulación de muestras espermáticas sometidas a un protocolo de criopreservación estandarizado con el fin de ver la influencia que estas etapas tienen en las características cinéticas de las células. Este estudio es importante debido a que, de manera general, el parámetro de motilidad espermática se ha tomado como referencia para la determinación de la fertilidad y calidad de muestras seminales, tanto en eyaculados frescos como en semen criopreservado (Henning et al 2014). En este caso, se ha llevado a cabo un análisis PCA seguido de un análisis de conglomerados para determinar la presencia de subpoblaciones espermáticas basados en diferentes parámetros de velocidad y linealidad espermática en diferentes etapas (fresco, 1ª centrifugación, 2ª centrifugación, enfriamiento o "cooling" y pos-descongelación).…”

“…Es necesario remarcar, que no sólo el número de subpoblaciones espermáticas varió notablemente entre las etapas estudiadas, sino que, además, debido probablemente a los diferentes medios utilizados y al sometimiento de las muestras espermáticas a diferentes temperaturas durante el proceso de criopreservación, la distribución de esas muestras espermáticas cambió radicalmente entre las diferentes etapas. Una posible explicación podría estar basada en los diferentes niveles de calidad espermática entre las diferentes subpoblaciones (Sbp1 a Sbp 4) derivado del cambio sufrido por los parámetros cinéticos medidos mediante sistema CASA (Henning et al, 2014). Además, las diferencias obtenidas en cuanto a los parámetros cinéticos en las diferentes etapas pueden deberse a diferentes estados fisiológicos de las células tales como el grado de capacitación espermática, el cual se lleva a cabo de una forma heterogénea y progresiva en las diferentes poblaciones celulares (García Herreros et al, 2005).…”

Investigation of in vitro measurable sperm attributes and their influence on electroejaculated bull semen with a fixed‐time artificial insemination protocol in AustralianBos indicuscattle

“…The proportion of progressively motile spermatozoa is one of the focal parameters for subjective semen quality assessment with the generalized assumption that the "faster the better." CASA offers a qualitative measure of motility patterns in the sperm trajectories (Abaigar, Cano, Pickard, & Holt, 2001;Henning, Petrunkina, Harrison, & Waberski, 2014;Satake et al, 2006;Sellem et al, 2015) and further examine the motile subpopulations of spermatozoa. For the chilled-stored semen for 48 hr, a significantly faster overall velocity, particularly along the curvilinear trajectory, was detected compared to the cryopreserved samples.…”

Effect of sexual steroids on boar kinematic sperm subpopulations

“…Boar ejaculates are diluted in extender and refrigerated before their use in artificial insemination. Once sperm leave the environment of the extender and enter the fluids of the female genital tract, these fluids modulate the motility dynamics of spermatozoa during their journey along the uterus and oviduct . The presence of sexual steroids along female reproductive tract has been demonstrated by the presence of receptors for progesterone , androgens , and estradiol .…”