Cat epididymal semen cryopreserved with and without vitamin E: effect on sperm parameters and lipid peroxidation

The aims of this study were to investigate: 1) if the addition of \u3b1-tocopherol (vitamin E) in three concentrations (0.3, 0.6 and 0.9 mM) is able to preserve spermatozoa integrity after thawing and 2) the effect of \u3b1-tocopherol supplementation on lipid peroxidation. Fifty four domestic cats were used in this study constituting 18 pools (3 cats per pool). Each pool was submitted at four experimental groups: group 0 (control) \u2013 epididymal sperm were frozen with a commercial Botucrio\uae extender; group 0.3, group 0.6 and group 0.9 \u2013 the extender was supplemented with 0.3, 0.6 and 0.9 mM of \u3b1-tocopherol, respectively. Each semen sample was evaluated for motility, progressive forward motility, morphology, sperm viability (plasma membrane integrity-PMI), hypo-osmotic swelling test (HOST), before and after thawing. The evaluation of lipid peroxidation reaction by Thiobarbituric Acid Reactive Substances (TBARS) test was performed on thawed semen only. Results demonstrated that there was no significant difference between control and the three \u3b1-tocopherol groups with regards to motility and progressive motility after thawing (P > 0.05). As expected, in fresh samples viability was significantly higher than in all the cryopreserved groups in which there was no positive influence of any of the \u3b1-tocopherol concentration used. Lipid peroxidation was higher in the supplemented groups 0.6 and 0.9 mM of \u3b1-tocopherol than in control and in 0.3 mM group. In conclusion, the addition of \u3b1-tocopherol to the commercial extender had no positive influence on reduction of lipid peroxidation. This topic deserves further investigations to better understand the effect of cryopreservation procedures on epididymal spermatozoa and to establish adequate strategies to counteract sperm cryodamages

Chemical imaging of canine oviducts during the post-ovulatory period

It is well known that bitches present a peculiar oocyte maturation, characterized by ovulation of immature oocytes and a long period of viability in the oviduct. Thus, the oviduct in this species plays an essential role in oocyte maturation, in addition to fertilization and early stage embryo development. Different approaches have been used in order to identify the factors involved in each step (from resumption of meiosis through development of embryos) and recently a new technique was used for spatial identification of oviductal proteins in the domestic cat (1). With the purpose to contribute to the understanding of these factors, we applied MALDI imaging mass spectrometry (MALDI-IMS) to obtain protein profiling and imaging of canine oviducts. Reproductive tracts were collected from 4 bitches in estrus (cross-breed, 2 to 6 years old) undergoing routine ovariohysterectomy. Post-ovulatory period was confirmed by blood serum progesterone concentrations (P4 mean\ub1SD: 15.5\ub12.5) and ovarian morphology. The oviducts were carefully dissected, divided into three segments (distal to the ovary-isthmus, proximal to the ovary-infundibulum and the mid-section between the two-ampulla; further confirmed by histology), snap-frozen in liquid nitrogen and stored at -80\ubaC until use. Then, they were sectioned (11 \ub5m) in a cryostat and fixed on ITO (indium tin oxide) conductive glass slides, while serial sections were collected on microscope slides for haematoxylin and eosin staining. For MALDI-IMS, samples were coated with a thin homogeneous layer of CHCA (\u3b1-Cyano-4-hydroxycinnamic acid) matrix using a nebulization device. MALDI images were acquired on an Autoflex III Smartbeam instrument (Bruker Daltonics) with 400 shots/spectrum, over the mass range of m/z 2 to 20 kDa in the positive ion mode. The spatial resolution of images was 80 \ub5m. Mass spectra were characterized by abundant ions of m/z 2279, 2401, 3458 and 4976; which have been tentatively attributed to actin cytoplasmic 2, keratin type 1, neutrophil defensin 1 and thymosin \u3b24, respectively. Actin has been detected in oviduct fluid of alpaca (2) and is related to epithelial cell renewal or secretory activity (3). As previously described in queens (1), defensin, keratin and thymosins are defense proteins that integrate the innate immune systems and are involved in the biological response to cellular damage. These data might contribute to piecing together the puzzle of factors that are involved in the peculiar aspects of the domestic dog reproductive physiology that might hamper in vitro embryo production. 1) Apparicio M, Santos VG, Rocha DFO et al. Matrix-assisted laser desorption/ionization imaging mass spectrometry for the spatial location of feline oviductal proteins. Reprod Domest Anim. 2017;52 (Suppl. 2):88\u201392. (2) Apichela SA, Arga\uf1araz ME, Zampini R et al. Biochemical composition and protein profile of alpaca (Vicugna pacus) oviductal fluid. Anim Reprod Sci. 2015,154:79-85 (3) Steffl M, Schweiger M, Sugiyama T et al. Review of apoptotic and non-apoptotic events in non-ciliated cells of the mammalian oviduct. Ann Anat. 2008,190: 46-52