title: Carnitine supplementation decreases capacitation-like changes of frozen-thawed buffalo spermatozoa.
abstract: The aim of this study was to evaluate the effect of carnitine supplementation of semen extender on fertility parameters of frozen-thawed buffalo sperm. Buffalo semen was cryopreserved in BioXcell containing 0 (control group), 2.5 and 7.5-mM carnitine. After thawing, viability, motility, membrane integrity and capacitation status (assessed by localization of phosphotyrosine-containing proteins and chlortetracycline, chlortetracycline assay) were evaluated. Furthermore, total antioxidant capacity, reactive oxygen species (ROS) and lipid peroxidation levels, as well as adenosine triphosphate (ATP) content and phospholipids concentration were assessed. Finally, in vitro-fertilizing ability was evaluated after heterologous IVF. An increased post-thawing sperm motility and membrane integrity were recorded in both treated groups compared with the control (44.4 ± 3.5, 53.1 ± 3.9, and 52.5 ± 3.6%; P < 0.05 and 48.44 ± 0.69, 55.19 ± 0.54, 59.63 ± 0.30%; P < 0.01 with 0, 2.5-mM, and 7.5-mM carnitine, respectively). Supplementation of carnitine to the freezing extender decreased (P < 0.01) the percentage of sperm displaying fluorescence at both equatorial and anterior acrosomal regions (pattern EA), corresponding to high capacitation level, compared with the control (30.3 ± 3.8, 18.8 ± 2.8, and 7.2 ± 2.9%, respectively, with 0, 2.5-mM, and 7.5-mM carnitine). In agreement with this, carnitine also decreased (P < 0.01) the percentage of sperm displaying chlortetracycline pattern B (capacitated sperm) (63.8 ± 1.8, 46.8 ± 2.2, and 37.2 ± 1.8%, respectively with 0, 2.5-, and 7.5-mM carnitine). Interestingly, carnitine increased total antioxidant capacity and ATP content of buffalo frozen-thawed sperm (1.32 ± 0.02, 1.34 ± 0.01, 1.37 ± 0.01 mM/L and 4.1 ± 0.1, 5.3 ± 0.1 and 8.2 ± 0.4 nM × 108sperm; P < 0.01, respectively, with 0, 2.5- and 7.5-mM carnitine). Intracellular ROS decreased in carnitine-treated sperm compared with the control, as indicated by dihydroethidium (DHE) values (0.22 ± 0.01, 0.18 ± 0.01, and 0.14 ± 0.0 μM/100 μL dihydroethidium, respectively, with 0, 2.5-, and 7.5-mM carnitine; P < 0.01), whereas lipid peroxidation levels (on average 30.5 ± 0.3 nmol/mL MDA) and phospholipids concentration (on average 0.14 ± 0.00 μg/120 × 106sperm) were unaffected. Despite the improved sperm quality, the percentage of normospermic penetration after IVF was not influenced (on average 53.5 ± 1.8). In conclusion, enrichment of extender with carnitine improved buffalo sperm quality by increasing ATP generation and modulating ROS production, without affecting in vitro fertilizing ability.
authors: Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.
authors: Valentina
authors: V
authors: Longobardi
authors: Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.
authors: Angela
authors: A
authors: Salzano
authors: Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy. Electronic address: giucampa@unina.it.
authors: Giuseppe
authors: G
authors: Campanile
authors: Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.
authors: Raffaele
authors: R
authors: Marrone
authors: Department of Political Science, Federico II University, Naples, Italy.
authors: Francesco
authors: F
authors: Palumbo
authors: Department of Biochemistry, Biophysics and General Pathology, II University of Naples, Naples, Italy.
authors: Milena
authors: M
authors: Vitiello
authors: Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.
authors: Gianluigi
authors: G
authors: Zullo
authors: Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.
authors: Bianca
authors: B
authors: Gasparrini
mesh_terms: D000818:Animals; D002020:Buffaloes; D002331:Carnitine / Q000494:pharmacology*; D002470:Cell Survival; D015925:Cryopreservation / Q000662:veterinary; D005615:Freezing; D008297:Male; D018384:Oxidative Stress; D012662:Semen Preservation / Q000662:veterinary*; D013075:Sperm Capacitation / Q000502:physiology*; D013081:Sperm Motility / Q000187:drug effects; D013094:Spermatozoa / Q000187:drug effects* / Q000502:physiology
chemical_list: D002331:Carnitine
keywords: ATP content; Buffalo sperm; Capacitation-like changes; Carnitine; Fertilizing ability; Oxidative stress