title: The utility of nanowater for ram semen cryopreservation.
abstract: Nanowater (NW; water declusterized in the low-temperature plasma reactor) has specific physicochemical properties that could increase semen viability after freezing and hence fertility after artificial insemination (AI) procedures. The main goal of this study was to evaluate ram semen quality after freezing in the media containing NW. Ejaculates from 10 rams were divided into two equal parts, diluted in a commercially available semen extender (Triladyl®; MiniTüb GmbH, Tiefenbach, Germany) prepared with deionized water (DW) or NW, and then frozen in liquid nitrogen. Semen samples were examined for sperm motility and morphology using the sperm class analyzer system and light microscopy. Cryo-scanning electron microscopy (cryo-SEM) was employed to determine the size of extracellular water crystals in frozen semen samples. Survival time at room temperature, aspartate aminotransferase (AspAT) and alkaline phosphatase (ALP) concentrations post-thawing as well as conception/lambing rates after laparoscopic intrauterine AI of 120 ewes were also determined. There were no significant differences between DW and NW groups in sperm progressive motility (26.4 ± 12.2 and 30.8 ± 12.4%) or survival time (266.6 ± 61.3 and 270.9 ± 76.7 min) after thawing and no differences in the percentages of spermatozoa with various morphological defects before or after freezing. There were, however, differences (P < 0.05) in AspAT (DW: 187.1 ± 160.4 vs. NW: 152.7 ± 118.3 U/l) and ALP concentrations (DW: 2198.3 ± 1810.5 vs. NW: 1612.1 ± 1144.8 U/l) in semen samples post-thawing. Extracellular water crystals were larger (P < 0.05) in ejaculates frozen in NW-containing media. Ultrasonographic examinations on day 40 post-AI revealed higher (P < 0.05) conception rates in ewes inseminated with NW (78.3%) compared with DW semen (58.3%), and the percentages of ewes that carried lambs to term were 73.3% and 45.0% in NW and DW groups, respectively (P < 0.01). In summary, the use of a semen extender prepared with NW was associated with a substantial improvement in the fertilizing ability of frozen-thawed ram semen and lamb productivity of inseminated ewes.
authors.affiliation: Department of Animal Biotechnology, University of Agriculture in Cracow, 30-274 Kraków, Poland.
authors.forename: Maciej
authors.initials: M
authors.lastname: Murawski
authors.affiliation: Institute of Animal Sciences, University of Agriculture in Cracow, 31-120 Kraków, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Schwarz
authors.affiliation: Department of Animal Biotechnology, University of Agriculture in Cracow, 30-274 Kraków, Poland joanna.grygier7@gmail.com.
authors.forename: Joanna
authors.initials: J
authors.lastname: Grygier
authors.affiliation: Department of Biology and Animal Breeding, University of Life Sciences in Lublin, 20-950 Lublin, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Patkowski
authors.affiliation: Nantes Nanotechnology Systems, 59-700 Bolesławiec, Poland.
authors.forename: Zdzisław
authors.initials: Z
authors.lastname: Oszczęda
authors.affiliation: Nantes Nanotechnology Systems, 59-700 Bolesławiec, Poland.
authors.forename: Igor
authors.initials: I
authors.lastname: Jelkin
authors.affiliation: Department of Animal Biotechnology, University of Agriculture in Cracow, 30-274 Kraków, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Kosiek
authors.affiliation: Department of Biology and Animal Breeding, University of Life Sciences in Lublin, 20-950 Lublin, Poland.
authors.forename: Tomasz M
authors.initials: TM
authors.lastname: Gruszecki
authors.affiliation: Department of Biology and Animal Breeding, University of Life Sciences in Lublin, 20-950 Lublin, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Szymanowska
authors.affiliation: Center for Interdisciplinary Research, Catholic University of Lublin, 20-705 Lublin, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Skrzypek
authors.affiliation: Department of Animal Biotechnology, University of Agriculture in Cracow, 30-274 Kraków, Poland.
authors.forename: Dorota A
authors.initials: DA
authors.lastname: Zieba
authors.affiliation: Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada N1G 2W1.
authors.forename: Pawel M
authors.initials: PM
authors.lastname: Bartlewski
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D001219:Aspartate Aminotransferases / Q000378:metabolism; D020285:Cryoelectron Microscopy; D015925:Cryopreservation; D005260:Female; D005307:Fertilization in Vitro; D008297:Male; D036103:Nanotechnology; D011247:Pregnancy; D018873:Pregnancy Rate; D012661:Semen / Q000201:enzymology; D012756:Sheep; D014867:Water
chemical_list: D014867:Water; D001219:Aspartate Aminotransferases; D000469:Alkaline Phosphatase
keywords: Cryopreservation; freezing; nanowater; ram; semen; sheep