LOW-DENSITY LIPOPROTEIN (LDL) SUPPLEMENTATION TO TRIS-BASED DILUENT AND ITS INFLUENCE ON VITAL AND ULTRA-STRUCTURAL CHARACTERISTICS OF FROZEN-THAWED CANINE SPERMATOZOA
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Date
2021
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Abstract
The present research was aimed to study the effect of low-density
lipoprotein (LDL) based extender on semen parameters like progressive motility.
viability, plasma membrane integrity, acrosome membrane integrity, DNA
integrity and mitochondrial membrane potential including ultrastructural
characteristics
Semen was collected from the dogs with a body condition score of 4-5 (on
a scale of 0-9) by digital manipulation. The pre-freeze characteristics of the semen
like volume, colour, consistency, concentration, mass motility, progressive
motility, viability and total sperm abnormalities were studied. Following which
the sperm rich fractions were diluted in the ratio of 1:1 or 1:2 with a TRJS-glucose
bound extender containing 20 per cent egg yolk in trial 1 and with TRIS-glucose
bound extender containing 8 per cent low-density lipoprotein (LDL) in trial 2
depending upon the sperm concentration, LDL was extracted as per the procedure
described by Moussa et al., (2002). Then cryopreservation was done as per
conventional method. The effect of LDL supplementation on progressive motility,
viability, plasma membrane integrity, acrosome membrane integrity, DNA
integrity and mitochondrial membrane potential at 24 h post-thaw were evaluated
and compared with Tris-egg yolk extender.
Morphological and functional characteristics of spermatozoa with EY and
LDL extender at 6 h of equilibration were recorded and was found to be
significantly higher for the percentage of viability, plasma membrane and DNA
integrity and mitochondrial membrane potential. However, no significant
difference was observed for plasma membrane integrity in EY and LDL added
semen at 6 h of equilibration.
After cryopreservation the semen samples were evaluated for progressive
motility, viability, plasma membrane integrity, acrosomal integrity, DNA integrity
and mitochondrial membrane potential at 24 hours post thaw and significantly
higher difference (p<0.01) was observed in the mean percentage of progressive
motility, viability, plasma membrane integrity, acrosomal integrity and
mitochondrial membrane potential. No significant difference was noticed in DNA
integrity between the semen extended with EY and LDL at 24 h post-thaw, which
might be due to more stable nuclear packing and highly condensed with a unique
DNA organization.
The semen samples diluted with EY and LDL extenders were evaluated
for all the above said parameters at 6 h of equilibration and 24 hours post thaw
and the result revealed that all the parameters were highly significant between 6 h
equilibration and 24 h post-thaw in the semen samples extended with both EY and
LDL. This could be because cryopreservation induces lethal intra-cellular ice
crystal formation in turn causing damage in the plasma and acrosomal membrane.
Cryopreservation also modifies the lipid biochemical organization and pattern. It
also changes the biochemical composition and structure of the spermatozoal
plasma and acrosomal membrane.
Ultrastructural characteristics of fresh semen, semen samples at 6 h of
equilibration and 24 h post-thaw by SEM showed that, there was more damage
noticed in different sperm regions in samples extended with EY at 6 h and 24 h
post-thaw when compared to LDL.
TEM micrograph of the canine semen added with EY extender showed
damaged acrosome and neck region while the semen extended with LDL showed
intact acrosomal membrane and intact head region with some damages inflicted in
the plasma membrane. TEM picture also revealed damaged acrosome with
leakage of enzymes and severe vesiculations and damage of the plasma membrane
in semen extended with EY while in semen added with LDL extender, head and
mid-piece were intact in TEM micrograph.
In conclusion, addition of LDL at 8 per cent concentration in Tris-glucose
bound extender could be a better alternative to egg yolk for cryopreservation of
canine semen as morphological, functional and ultra-structural damages induced
by cryopreservation are minimal.