Optimizing conditions for treating goat semen with cholesterol-loaded cyclodextrins prior to freezing to improve cryosurvival q C. Konyali a,1 , C. Tomás a , E. Blanch a , E.A. Gómez a , J.K. Graham b , E. Mocé a,⇑ a Centro de Tecnología Animal-Instituto Valenciano de Investigaciones Agrarias (CITA-IVIA), Apdo 187, 12400 Segorbe, Castellón, Spain b Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA article info Article history: Received 8 October 2012 Accepted 3 June 2013 Available online 11 June 2013 Keywords: Semen Frozen–thawed sperm Freezing Goat Caprine abstract The fertility of goat sperm is highly variable and new methods for improving sperm cryosurvival are needed. Cholesterol plays important roles in membrane fluidity, cold shock sensitivity and cryodamage, and treating sperm from cold-shock sensitive species with cholesterol-loaded cyclodextrins (CLC) prior to cryopreservation enhances sperm cryosurvival. The aim of this study was to develop a CLC-treatment to optimize goat sperm cryopreservation. A total of 45 ejaculates coming from eleven adult Murciano-Gra- nadina bucks were used and three experiments were conducted to determine: (1) the optimal CLC con- centration to treat goat sperm; (2) the optimal time to treat the sperm (before or after seminal plasma removal); and (3) optimal freezing diluent (either of two Tris-citrate diluents containing 2% or 20% egg yolk and 4% glycerol or a skim milk diluent with 7% glycerol) to cryopreserve goat sperm. Goat sperm cryosurvival rates were greatest when they were treated with 1 mg CLC/120  10 6 sperm prior to freez- ing. The benefit was also greatest if the sperm were treated with CLC after seminal plasma removal. Finally, CLC treatment improved sperm cryosurvival rates for sperm frozen in all three diluents, however, CLC treatment was most effective for sperm frozen in egg-yolk diluents. In conclusion, treating goat sperm, with CLC prior to cryopreservation, improved sperm cryosurvival rates. In addition, CLC treatment was effective for all freezing diluents tested, making this technology practical for the industry using cur- rent cryopreservation techniques. Nevertheless, additional studies should be conducted to determine how CLC might affect sperm functionality and fertilizing ability. Ó 2013 Elsevier Inc. All rights reserved. Introduction The recent surge in centers specializing in providing reproduc- tive technologies, including custom semen collection, cryopreser- vation and artificial insemination, for livestock species, necessitates developing cryopreservation protocols specific for each species. Semen cryopreservation is a valuable technique for goat production, since it permits collecting and storing semen out- side of the breeding season, for use later, which can increase the number of females that can be inseminated with sperm from a spe- cific male. In addition, semen can be transported to distant areas and can be used as insurance against injury or even death of a genetically valuable male. Therefore, cryopreserved sperm can eco- nomically impact the commercial livestock production for several species. In spite of the great advantages associated with using cryopreserved sperm, it is still not used extensively on commercial goat farms. Cryopreservation can have detrimental effects on sperm quality [13,24,25,40,46], including a loss of sperm motility, induction of structural and functional changes in sperm, and sperm death. These detrimental effects can reduce the fertilizing ability of sperm, even though the cell may survive cryopreservation. There- fore, the kindling rate of fresh goat sperm is estimated to be approximately 12.1% higher than that observed for frozen–thawed sperm [26]. In addition, the fertility of frozen–thawed goat sperm ranges from 3% to 70% [18], and this disparity seen in the cryosur- vival rates of goat semen is a significant reason that frozen–thawed goat semen is not widely used on commercial goat farms [5]. The cryosurvival rates of sperm, from the various species, can be very different. Some of the species differences observed can be 0011-2240/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cryobiol.2013.06.001 q Statement of funding: C. Konyali received a scholarship from CIHEAM (Zaragoza, Spain), C. Tomás was supported by a scholarship from Ministerio de Ciencia e Innovación (Ref. BES-2007-17063; Madrid, Spain) and E. Blanch was supported by a scholarship from Conselleria d’Agricultura, Pesca y Alimentació (CAPA, DOGV5324; Valencia, Spain). E. Mocé was supported by funds from INIA-CCAA and the European Social Fund (Ref. DR03-704) and is currently supported by funds from Ministerio de Ciencia e Innovación from Subprograma Ramón y Cajal (Ref. RYC-2010-06162; Madrid, Spain). ⇑ Corresponding author. Fax: +34 964 710218. E-mail addresses: ckonyali@gmail.com (C. Konyali), critoal@hotmail.com (C. Tomás), blantor@hotmail.com (E. Blanch), gomez_ern@gva.es (E.A. Gómez), James.K.Graham@ColoState.EDU (J.K. Graham), moce_eva@gva.es (E. Mocé). 1 Present address: Canakkale Onsekiz Mart University, Lapseki Vocational College, 17800 Canakkale, Turkey. Cryobiology 67 (2013) 124–131 Contents lists available at SciVerse ScienceDirect Cryobiology journal homepage: www.elsevier.com/locate/ycryo