Effect of isolation protocols and cryoprotectants on freezing of stallion epididymal spermatozoa.

BACKGROUND: The recovery of spermatozoa from the cauda epididymis may be the only option to obtain genetic material from elite stallions that had undergone castration or sudden death due to colic or severe injury. OBJECTIVE: To evaluate two different protocols for retrieval of stallion epididymal spermatozoa and to evaluate different cryoprotectants on the freezability of the epididymal spermatozoa. MATERIALS AND METHODS: Six epididymides from three stallions were collected immediately after routine castration under general anesthesia. In the first experiment, each epididymis (of two testes) of the same stallion were processed using different methods for retrieval of the epididymal spermatozoa and were pooled and cryopreserved either using 5% glycerol or 5% dimethyl formamide (DMF) as cryoprotectant. The semen quality parameters viz., progressive motility, HOST, viability and acrosome integrity were evaluated at the fresh, pre-freeze and post-thaw stages. RESULTS: Retrograde method of flushing of epididymis yielded significantly (p < 0.05) higher concentration of the stallion sperm than that of the floating method. The qualitative semen parameters i.e., viability, plasma membrane integrity and acrosome integrity were found to be significantly restored using 5% DMF as cryoprotectant in comparison to when 5% glycerol was used. CONCLUSION: Retrograde flushing method of epididymis yielded significantly higher sperm concentration to that of the floating method, and 5% DMF as cryoprotectant provided acceptable freezability of stallion epididymal spermatozoa. DOI: 10.54680/fr23310110312.

Microwave assisted fast fabrication of zinc/iron oxides based polymeric nanocomposites and evaluation on equine fibroblasts.

We aimed to provide a tissue repair material, which can be synthesized rapidly, using polymers mimicking the natural environment in the extra-cellular matrix and metals/minerals. The components should have the potential to be used in tissue repair and simultaneously, reducing the side-effects of the incorporated molecules. It is challenging to manage the dispersibility of ZnO NPs in common solutions like water. Here, we report a novel method for preparing highly dispersible suspensions of ZnO NPs. In contrast to those synthesized by conventional methods, microwave assisted method allowed synthesis of dispersible ZnO NPs and the incorporation of zinc/Iron oxides NPs within alginate and gum matrix (AG) in a short span of time providing high yield of the product. The nanoformulations were characterized for size, morphology, interaction of various chemicals used during their synthesis by transmissible electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and energy dispersive X ray Spectrum. It was also evaluated for cytotoxicity and their effect on equine fibroblast cells. Microwave-assisted fabrication of zinc/iron oxides nanoparticles provided flowerlike morphology with good dispersibility and high yield in a short span of time. Our results revealed that ZnO NPs were more cytotoxic than AG ZnO NPs and doped AG Fe3O4 doped ZnO NPs at higher concentrations. Further metal nanoparticles capped with alginate/acacia with size range less than 100 nm demonstrated high stability, good biocompatibility, re-epithelization and enhanced mineralization in horse fibroblast cells.

Effects of different concentration and combinations of cryoprotectants on sperm quality, functional integrity in three Indian horse breeds.

Traditionally Glycerol (Gly) is being used as major cryoprotectant and its toxicity could be a reason for the variation on stallion sperm freezability and fertility. In an effort to minimize Gly toxicity alternative cryoprotective agents like Di-methyl Formamide (DMF) have been investigated. The effect of the cryoprotectant and dose of cryoprotective agent varies from breed to breed and also from stallion to stallion within the same breed. Considering these factors a study was designed to study the effects of Gly and DMF at different concentrations and combinations on the plasma membrane, acrosome and DNA integrity as well as other post thaw seminal characteristics of semen of three Indigenous stallion breeds. In the current study, semen was collected from apparently healthy 4-6 years old 3 Marwari, 3 Manipuri and 3 Zanskari breed stallions. After semen collection and evaluation of fresh semen, each semen sample was extended with semen extender containing different concentrations and combinations of Gly and DMF cryoprotectants (i.e. 5% Gly, 5% DMF, 2% Gly, 2% DMF, 2.5% Gly +2.5% DMF and 1% Gly +1% DMF) and frozen. Post thaw semen evaluation was done on the basis of post thaw motility, live sperm count, hypo osmotic swelling test, acrosomal integrity and DNA integrity. Frozen thawed semen showed that the values of plasma membrane integrity, acrosome integrity and DNA integrity parameters were significantly higher (P < 0.05) with 5% DMF than the other cryoprotectants levels and combinations of Gly and DMF. From the present study, it was inferred that the combination of cryoprotectants at different concentrations (Gly and DMF @ 2.5 and 1%) also could not show better enhancement compared to the single cryoprotectant i.e DMF @5% in various post thaw seminal characteristics of Indigenous stallion semen. DMF at 5% concentration gave better protection to the plasma membrane and retained the acrosome and DNA integrity of the spermatozoa. Hence it can be concluded that DMF at 5% can be used for the cryopreservation of the Indigenous stallion with better preservation of the seminal quality.