The cryopreservation of equine semen, although advantageous, has critical points: the spermatozoa undergo structural and
physico-chemical changes which cause alterations of flagellum,
plasma membrane and cellular organelles, compromising the fertilizing potential (Freitas et al. J Equine Vet Sci. 2016; 46: 1–6).
The aim of the work was to evaluate whether the post-thawing
addition of relaxin, a pleiotropic hormone, had positive effects
on motility, velocity, viability, membrane and DNA integrity, mitochondrial activity and apoptosis rate of the equine semen. The
data were compared with a previous work, in which relaxin had
been added before cryopreservation to the same semen batches
(Elkhawagah et al. Anim Reprod Sci. 2020; 106351). The semen
of 3 stallions of proven fertility was collected, pooled, and frozen
using standard procedures, with relaxin supplementation (0, 12.5,
25, 50, 100 ng/ml). In this work, relaxin was added after thawing to the row semen at the same concentrations. Immediately after the addition of relaxin and after 15’, 30
, 60
, 90 and 120 of
incubation (37°C, CO2 5%), sperm motility and velocity were evaluated with CASA system, viability was assessed through Hancok test
and membrane integrity with Hypoosmotic Swelling test. DNA integrity, mitochondrial activity and apoptosis rate were assessed by
acridine orange staining, JC-1 and annexin V-PI (flow cytometric
analyses). Viability, mitochondrial activity, DNA integrity and percentage of apoptotic spermatozoa were not significantly improved
after post-thawing addition of relaxin, but it seemed to exert
a positive effect on the mitochondrial membrane potential and
on the prevention of apoptosis, in accordance with Ferlin et al.
(J Androl. 2012; 33: 474–482). The percentage of spermatozoa
with intact membrane significantly increased in the samples
with the post-thawing addition of 12.5, 25 and 50 ng/ml relaxin,
but not in those supplemented with relaxin before freezing. The
post-thawing addition of 50 ng/ml relaxin implemented and
maintained sperm motility and velocity over time, particularly
after 90 min of incubation, and the same results were obtained
after 30 minutes of pre-freeze relaxin supplementation at 12.5
ng/ml. It was concluded that one protocol was no better than
the other, but the pre-freeze addition of relaxin is more suitable for classical AI, while the post-freeze supplementation is
better for deep AI, endoscopic AI or ICSI, as it also seems to
activate capacitation. In the future, establishing the metabolic
pathways through which relaxin acts on the male gamete will
be essential to fully exploit the potential of a hormone that,
only recently, has been found to be extremely important for male
fertility |
