title: Cholesterol-loaded cyclodextrins prevent cryocapacitation damages in buffalo (Bubalus bubalis) cryopreserved sperm.
abstract: The aim of this study was to investigate the effect of cholesterol-loaded cyclodextrins (CLC) on motility, viability, capacitation status, and in vivo fertility of buffalo frozen-thawed sperm. After the initial semen assessment, buffalo sperm were diluted in BULLXcell extender containing 0- (control), 1.5-, and 3-mg/mL CLC and cryopreserved. At thawing, sperm motility was evaluated by phase contrast microscopy, and viability-capacitation status was assessed by Hoechst 33258-chlortetracycline (CTC) assay. Capacitation status was also evaluated by an indirect immunofluorescence assay to localize phosphotyrosine-containing proteins. Moreover, buffaloes were artificial inseminated to assess the in vivo-fertilizing potential of CLC-treated semen. No differences among control, 1.5-, and 3-mg/mL CLC-treated groups were recorded in both sperm motility (66.5 ± 5.6, 68.8 ± 4.8, and 68.8 ± 4.8, respectively) and viability (86.5 ± 1.9, 87.6 ± 1.5, 88.4 ± 2.3, respectively). However, the extender supplementation with CLC significantly reduced sperm cryocapacitation. Indeed, CLC treatment decreased (P < 0.01) the proportion of sperm showing the CTC pattern B (capacitated sperm) compared with the control (69.6 ± 3.4, 37.8 ± 1.5, and 51.3 ± 4.7, respectively, with 0, 1.5-, and 3-mg/mL CLC; P < 0.01). Furthermore, the percentage of sperm displaying tyrosine-phosphorylated pattern EA (i.e. high capacitation level) was reduced (P < 0.01) in both CLC-treated groups (10.8 ± 3.3 and 5.6 ± 1.6, respectively, with 1.5- and 3-mg/mL CLC) compared with the control (37.3 ± 6.9), reaching values similar to those recorded in fresh semen (11.0 ± 3.5). In addition, treating sperm with 3-mg/mL CLC increased (P < 0.01) the percentage of nonfluorescent (pattern NF), i.e., non-capacitated sperm (41.8 ± 3.6) compared with fresh semen (11.0 ± 6.9). No differences were recorded in pregnancy rates at 60 days post-artificial insemination among control, 1.5- and 3-mg/mL CLC groups (59.7%, 65.6%, and 56.9%, respectively). In conclusion, CLC treatment of buffalo sperm strongly decreases sperm cryocapacitation damages, without affecting the in vivo fertilizing capability.
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: Giuseppe
authors: G
authors: Albero
authors: Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.
authors: Carolina
authors: C
authors: De Canditiis
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.
authors: Antonio
authors: A
authors: Natale
authors: Istituto Zooprofilattico Sperimentale del Mezzogiorno, Portici, Naples, Italy.
authors: Anna
authors: A
authors: Balestrieri
authors: Department of Veterinary Medicine and Animal Production, Federico II University, Naples, Italy.
authors: Gianluca
authors: G
authors: Neglia
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: Bianca
authors: B
authors: Gasparrini
mesh_terms: D000818:Animals; D002020:Buffaloes; D002784:Cholesterol / Q000494:pharmacology*; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D003505:Cyclodextrins / Q000494:pharmacology*; D007315:Insemination, Artificial / Q000662:veterinary; D008297:Male; D055101:Semen Analysis / Q000662:veterinary; D012662:Semen Preservation / Q000379:methods / Q000662:veterinary*; D013075:Sperm Capacitation; D013094:Spermatozoa / Q000187:drug effects* / Q000502:physiology
chemical_list: D003505:Cyclodextrins; D002784:Cholesterol
keywords: Buffalo; Cholesterol-loaded cyclodextrins; Cryocapacitation; Fertilizing ability; Sperm