Arunmozhi NRajput Shubhamsingh ChandansinghTANUVASRangasamy SKannan TA2023-10-252023-10-252021https://krishikosh.egranth.ac.in/handle/1/5810199662The 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.EnglishThesis