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Available online at www.sciencedirect.com 2) 184 009 httpDonkey jack (Equus asinus) semen cryopreservation: Studies of seminal parameters, post breeding inflammatory response, and fertility in donkey jennies
A. Rota*, D. Panzani, C. Sabatini, F. Camillo
Dipartimento di Clinica Veterinaria, Università di Pisa, Pisa, Italy
Received 19 September 2011; received in revised form 3 July 2012; accepted 15 July 2012 stract
The aims of this study were (1) to evaluate motility parameters of donkey jack (jack; Equus asinus) semen cryopreserved in INRA-96
RA; IMV Technologies, France, 2% egg-yolk enriched) using either glycerol (GLY) or ethylene glycol (EG) as a cryoprotector; (2) compare in vitro the postthaw re-extension with homologous seminal plasma (SPL) or INRA; (3) to compare fertility in donkey nies (jennies; Equus asinus) timed artificially inseminated with jack semen cryopreserved using GLY or EG, re-extended with INRA; to compare fertility in jennies timed artificially inseminated with jack semen cryopreserved using GLY re-extended with SPL, INRA, not re-extended (NN); and (5) to describe some preliminary results of the inflammatory uterine response postbreeding. Semen from jacks was collected and frozen in an INRA-2% egg yolk extender added of either 2.2% GLY or 1.4% EG. Postthaw motility was luated by a computer-assisted motility analyzer. Uterine inflammatory response and fertility were evaluated after artificial insemiion (AI) of 13 jennies with frozen-thawed semen, either further extended with INRA (Group GLY-INRA, 13 cycles, and EG-INRA, ycles), or with SPL (Group GLY-SPL, 13 cycles), or not re-extended (GLY-NN, 5 cycles). In each cycle, jennies were bred twice h 500 106 sperm cells (250 106 from each jack), at fixed times after induction of ovulation, and uterus was flushed at 6 and h after first and second breeding, respectively. Cells in the recovered fluid were counted and distinguished as polymorphonuclear trophils (PMN) or other cell types. Total and progressive motility did not differ between cryoprotectants, but were higher when en samples were re-extended in INRA, compared with SPL (P 0.05). Pregnancy was diagnosed by transrectal palpation and asonography examinations at 14 and 16 days postovulation. In 7/13 (53.8%) jennies and 12/39 (30.4%) cycles postbreeding auterine fluid accumulation was observed, with no differences between treatments (P 0.05). Polymorphonuclear neutrophil bers and concentrations were higher in the first flushing compared with the second, and PMN concentration was higher in GLY-SPL n in GLY-INRA (P 0.05). Pregnancy rates in GLY-SPL, GLY-INRA, EG-INRA, and GLY-NN were 8/13, 3/13, 2/8, and 1/5, pectively. There was no significant difference either between the two cryoprotectants re-extended in INRA, or between re-extension ups. There was however a trend for GLY-SPL to improve pregnancy rates compared with GLY-INRA (P 0.055). These results icate that it is possible to obtain similar postthaw sperm motility and pregnancy rates using GLY or EG as a cryoprotectant for donkey en, and that in the conditions of this study the re-extension in SPL of thawed semen before AI showed a trend toward the provement of fertility and increased PMN concentration in uterine flushings. 2012 Elsevier Inc. All rights reserved. words: Donkey; Semen cryopreservation; Seminal plasma; Postmating endometritis; Fertility 1. IntroductionTheriogenology 78 (201the
Corresponding author. Tel.: 390502210163; fax 390502210182.
E-mail address: firstname.lastname@example.org (A. Rota). 3-691X/$ – see front matter © 2012 Elsevier Inc. All rights reserved. ://dx.doi.org/10.1016/j.theriogenology.2012.07.0156–1854 www.theriojournal.comConsidering the importance of biodiversity and of preservation of domestic species genetic resources, the (E ori can to ma str at
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SP po po the wo cry the im 1847A. Rota et al. / Theriogenology 78 (2012) 1846–1854creation of genetic banks for endangered donkey quus asinus) breeds is needed. The Amiata donkey, ginating from the homonymous mountain in Tusy, Italy, is a medium size breed (withers height 125 130 cm) of working animals, with characteristic rkings: dark gray coat, shoulder stripe, and zebra ipes in the legs . This breed was declared a breed risk of extinction by the Biodiversity Committee of ropean Parlament (NL 215/90) and by the Food and riculture Organization of the United Nations . To te, artificial insemination (AI) with donkey cryopreved semen has given disappointing results [3–5]. It s been hypothesized that glycerol (GLY), the cryotectant most commonly used for horse (Equus callus) semen cryopreservation, may be toxic for dony jack (jack) spermatozoa  or exert a negative ect on donkey jenny (jenny) fertility . The hythesis of an effect of this compound on the jenny nital tract rather than on the sperm cell seems to be ported by the higher pregnancy rates observed in rse mares (mares) compared with jennies after fro-thawed donkey semen AIs . When GLY was d as cryoprotectant, jack frozen-thawed semen alys resulted in satisfactory pregnancies rates in res, varying from 38% to 40% [2,3] to 50% to 53% ; while pregnancy rates per cycle were poor (0 to %) when jennies were bred [3–5]. Other cryoprotants have thus been tested (dimethyl sulfoxide, dithyl formamide, dimethyl acetamide) but pregnancy es in jennies ranged from 0  to 11% (5). The use ethylene glycol (EG) for jack semen preservation ulted in a pregnancy rate of 43% in mares . To our owledge, jenny fertility after AIs of jack semen cryoserved with this molecule has not yet been evalud.