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Current study was aimed to assess the β-cyclodextrin (βCD) and methyl-β-cyclodextrin (MβCD) on delivery of cholesterol, and substitution of fructose and glycerol with trehalose on the ram semen cryosurvival. Samples were collected, diluted with Tris-citric acid-LDL extender, pooled, and used. In experiment I, βCD and MβCD carriers were used and compared to deliver the cholesterol (at 0, 0.5, 1, 1.5, 2, and 4 mg/mL). In the experiment II, trehalose (0, 7, 14, 21, and 28 mM) was substituted with fructose (28, 21, 14, 7, 0 mM, respectively). In the experiment III, the best cholesterol/carrier dose groups from the first experiment, were selected to be evaluated with the fructose/trehalose (14/14 mM) combination compared to fructose (28 mM) alone. The concentration of glycerol in the above-mentioned experiments was set at 4.5%. In the experiment IV, the effect of lowering glycerol (4% vs. 4.5%) was assessed using selected cholesterol/carrier groups. Kinematics, chromatin integrity, ATP contents, malondialdehyde amounts and viability were evaluated. Cholesterol (especially at 1.5 and 2 mg/mL) improved the kinematics and ATP levels using both carriers. The optimised amounts of trehalose (14 mM)/fructose(14 mM) reduced peroxidation and DNA fragmentation levels. Co-administration of optimised levels of cholesterol with trehalose/fructose did not show extra beneficial effects compared to each of them. Trehalose could not protect the spermatozoa at lower amounts of glycerol (4% vs. 4.5%). In conclusion, either the optimised levels of cholesterol (using βCD or MβCD carriers) or substitution of half of the fructose with the trehalose alone could lead to improvement in quality of frozen/thawed ram semen., (© 2024 Wiley‐VCH GmbH. Published by John Wiley & Sons Ltd.)

Background: Wide range of ovulation distribution is the main restricting factor in establishing the pregnancy following oestrus synchronization (ES) and fixed time insemination (FTI) in sheep., Objectives: Determining the ovulation time (OVT) following ES with two different vaginal devices, its relation to progesterone and conception upon FTI with frozen/thawed semen., Methods: Oestrus was synchronized using either controlled internal drug release (CIDR) (ewe, n = 6; ewe lamb, n = 5) or vaginal sponge (ewe, n = 6; ewe lamb, n = 5) insertion for 12 days, plus Equine chorionic gonadotropin (eCG) at devices removal (Day 0). Sizes of the ovarian follicles were measured using transvaginal probe at -12, 0 and 30-33, 53 h and continued every 3-4 h until 75 h after eCG treatment. Serum progesterone amounts were measured at -12, 0, +2 and +11. Laparoscopic FTI was done at 60.5 ± 0.5 h., Results: The CIDR-treated group initiated and completed ovulations earlier compared to sponge-treated females (median: 64 vs. 71 h; p < 0.05). Ewe lambs were ovulated earlier compared to ewes in the sponge-treated group (66.71 vs. 71.5; p = 0.017). Mean sizes of ovulatory follicles and corpora lutea were not affected by device types. Higher amounts of progesterone were observed in CIDR group compared to sponge-treated group at device removal (2.68 ± 0.12 vs. 0.30 ± 0.01 ng/mL; p < 0.001). The conception was confirmed in 2/10, and 5/11 females of sponge and CIDR-treated females, respectively., Conclusions: Types of progestogens influence the OVT, and consequently the result of FTI with frozen/thawed semen. The optimum timespan for FTI should be chosen according to the device types during non-breeding season., (© 2023 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

The current study was designed to evaluate the effect of sequential low and high dietary linseed oil (LO; as omega-3 enriched fatty acid; FA) before and post insemination, respectively, on different plasma variables of ewes. Fat-tailed Qezel ewes were assigned randomly to be fed a diet enriched with 3% LO (n = 30) or the saturated FA (SFA; n = 30) three weeks before insemination (Day 0). The lipogenic diet supplemented with 6% LO or SFA was fed after insemination until Day +21. The control ewes were fed an isocaloric and isonitrogenous diet with no additional FA during the study. Estrus was synchronized by inserting a vaginal sponge (Spongavet ® ) for 12 days + 500 IU equine chorionic gonadotropin (eCG; Gonaser ® ), and ewes were inseminated via laparoscopic approach 56-59 h after eCG injection. The size of ovarian structures was assessed by transvaginal ultrasonography at -21, -14, -2, 0, and +10 days. Blood samples were collected weekly to measure the plasma's different biochemical variables and FA profile. Treatment did not affect the amounts of glucose, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, interleukin-10, interleukin-2, and non-esterified FA ( p > 0.05). Conversely, concentrations of triglyceride, cholesterol, tumor necrosis factor-alpha, and insulin-like growth factor-1 were higher in SFA-fed ewes relative to control animals ( p < 0.05). LO feeding resulted in greater amounts of n -3 FA isomers in plasma, while higher amounts of stearic acid were detected in SFA fed group 0 and +21 ( p < 0.05). The number of ovarian follicles and corpora lutea also were not affected by treatment. Other reproductive variables were not affected by treatment except for the reproductive rate. It seems that LO or SFA feeding of fat-tailed ewes peri-insemination period was not superior to the isocaloric non-additional fat diet provided for the control group during the non-breeding season., Competing Interests: No potential conflict of interest relevant to this article was reported., (© Copyright 2023 Korean Society of Animal Science and Technology.)

The main purpose of the current study was to find suitable and optimum levels of protectants among chicken egg yolk plasma (CEYP) and low-density lipoproteins (LDLs), alone or supplemented with ewe or cow skim milk, for cryopreservation of ram semen. In Experiments 1 and 2, the CEYP (28%) freezing extender was enriched with ewe or cow milk (2.5%, 5%, 10%, or 20%; v/v), respectively. In Experiments 3 and 4, the semen extender was prepared by varying the amounts of fresh or lyophilized LDL (lyo-LDL), respectively. Finally, ewe or cow skim milk was added to the freshly extracted LDL extender and the quality of frozen-thawed semen was examined (Experiments 5 and 6). Kinematics of spermatozoa (assessed using a computer-assisted sperm analysis system), viability, functionality of the plasma membrane, and levels of malondialdehyde (MDA) and total antioxidant capacity (TAC) were evaluated. Results revealed that addition of ewe or cow skim milk (5%, 10%, or 20%; v/v) to the CEYP diluent enhanced kinematics, viability, and membrane integrity of spermatozoa compared with the control ( p  < 0.05). Moreover, fresh LDL diluent was more effective than lyo-LDL in the cryosurvival of ram spermatozoa. In addition, enrichment of fresh LDL diluent with ewe or cow skim milk improved different variables of spermatozoa compared with the control ( p  < 0.05). Levels of MDA and TAC were not affected by adding ewe or cow milk to the diluents ( p  > 0.05). In conclusion, enrichment of fresh LDL extenders with ewe or cow milk also is proposed as an approach to preserve ram semen quality against cold shock and cryodamage injuries.

Background: Due to lower antioxidant capacity and higher amounts of polyunsaturated fatty acids, ram spermatozoa are very susceptible during cooling process., Objectives: The objective was to examine the effect of the trans-ferulic acid (t-FA) on the ram semen during liquid preservation., Methods: Semen samples were collected from the Qezel rams, pooled, and extended with the Tris-based diluent. Pooled samples enriched with different amounts of the t-FA (0, 2.5, 5, 10, and 25 mM) and preserved at 4°C for 72 h. Spermatozoa's kinematics, membrane functionality, and viability were assessed by CASA system, hypoosmotic swelling test, and eosin-nigrosin staining, respectively. Moreover, biochemical parameters were measured at 0, 24, 48, and 72 h., Results: Results showed that 5 and 10 mM t-FA improved forward progressive motility (FPM) and curvilinear velocity compared to the other groups at 72 h (p < 0.05). Samples treated with 25 mM t-FA showed the lowest total motility, FPM, and viability at 24, 48, and 72 h of storage (p < 0.05). Higher total antioxidant activity levels were observed in the 10 mM t-FA-treated group compared to the negative control at 72 h (p < 0.05). Treatment with 25 mM t-FA increased malondialdehyde amounts and decreased superoxide dismutase activity compared to other groups at the final time assessment (p < 0.05). Nitrate-nitrite and lipid hydroperoxides values were not affected by treatment., Conclusions: The current study indicates the positive and negative influences of different concentrations of t-FA on the ram semen upon cold storage., (© 2023 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

The current study was conducted to determine the optimal concentration carrier-compound for oleic acid (OA) among dimethyl sulfoxide (DMSO), liposome and β-cyclodextrin on ram spermatozoa cryosurvival. The preliminary experiment was designed to ascertain the optimal concentration of egg yolk plasma. In Experiment 1, semen was placed in a diluent containing different concentrations of OA dissolved in DMSO (0.125, 0.25, 0.50, 1, 2, 4 and 8 mM). In Experiments 2 and 3, effects of liposome loaded-OA and β-cyclodextrin-OA complexes (0.25, 0.50, 1 and 2 mM) on semen cryopreservation were evaluated. In Experiment 4, optimal concentrations of OA were determined, based on results from previous experiments. Spermatozoa viability, kinematics, plasma membrane integrity, malondialdehyde, superoxide dismutase activity and total antioxidant status of samples were evaluated. Results indicated varying concentrations of OA had different effects on sperm kinematics, viability and membrane functionality after freezing/thawing (P < 0.05). In addition, inclusion of OA in liposomes or combinations with β-cyclodextrin resulted in greater values for spermatozoa motion variables compared with DMSO dissolved-OA (P < 0.05). Inclusions of OA at 0.25 and 0.50 mM led to a reduction in amounts of lipid peroxidation when there was inclusion of liposome and β-cyclodextrin as carrier-compounds (P < 0.05). Activity of SOD was similar with inclusion of different concentrations of OA or carrier-compounds, but total antioxidant capacity was affected by OA concentration and carrier-compound type (P < 0.05). The results highlight the importance of carrier-compound type and concentrations of OA on ram spermatozoa during cryopreservation., Competing Interests: Declaration of Competing Interest None of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The current study was designed to investigate the effect of idebenone (Id), an antioxidant on ram semen quality. Semen samples were collected, pooled and diluted in a Tris-based extender supplemented with 0, 1, 2, 4 and 8 µM idebenone. Computer-assisted sperm analysis was used to evaluate spermatozoa kinematics. Sperm viability and membrane functionality were assessed respectively, by eosin-nigrosin staining and HOS test. Biochemical assays were carried out to measure different metabolites in spermatozoa and medium at 0, 24, 48 and 72 hr. Total and forward progressive motility were greater in 1, 2 and 4 µM idebenone treated groups compared to control at 24, 48 and 72 hr time points (p < 0.05). Semen supplementation with Id significantly increased viability and functionality of spermatozoa membrane during storage (p < 0.05). Lower amounts of lipid hydroperoxides in medium and spermatozoa were observed in Id-treated groups compared to control one at 24 and 48 hr of storage (p < 0.05). Medium and spermatozoa amounts of malondialdehyde and nitric oxide were less in Id 4 µM group compared to the control at 72 hr (p < 0.05). Total antioxidant capacity values and superoxide dismutase activity of spermatozoa and medium were greater in 2 and 4 µM idebenone treated groups in comparison with the control at 72 hr (p < 0.05). Results of the current study indicated that ram semen supplementation with Id at 4 µM level improved quality by ameliorating nitrosative and peroxidative stress, hence could be considered as an antioxidant additive during storage at 4°C., (© 2018 Blackwell Verlag GmbH.)

This study was aimed to investigate the effects of 17 𝛽-estradiol (E2) and 𝛼-zearalenol (α-ZOL) on motility parameters, plasma membrane integrity, levels of produced nitric oxide (NO) and total antioxidant capacity of Ghezel ram sperm during the liquid storage at 4 ˚C, for various periods of time. Semen samples were collected from four rams and diluted with Tris-egg yolk extender and supplemented with E2 (100 µmol) or different concentrations of α-ZOL (100 pmol, 100 nmol and 100 µmol) at a final concentration of 200 × 10 6 sperm per mL. We failed to show any significant effect of E2 at 100 µmol concentration on ram's sperm parameters while α-ZOL resulted in a significant decrease of plasma membrane integrity at 100 µmol concentration (55.40% for α-ZOL vs 62.20% for control) after 96 hr incubation. Alpha-ZOL had decreasing effect on sperm motility parameters including curvilinear velocity and average path velocity at 100 µmol concentration after 96 hr storage. Although remarkable reduction of total antioxidant capacity at high concentration of α-ZOL and long incubation time was found, however no significant changes were recorded in NO level during storage time. It was concluded that the detrimental effect of α-ZOL on ram sperm might be attributed to its induced oxidative stress and damage to the plasma membrane., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper.