Effect of Ultra-Rapid Cryopreservation on Sperm Head Morphometry of Wild Species
2018 Joint EAZWV/AAZV/Leibniz-IZW Conference
Eva Martínez-Nevado1, DVM; Emma O’Brien2, Dr. biol. sci., PhD; Milagros C. Esteso2, DVM, PhD; Adolfo Toledano-Díaz2, Dr. biol. sci., PhD; Cristina Castaño2, DVM; Paula Bóveda2, Dr. biol. sci.; Juncal García García1, DVM; Rafael Guerra3, DVM; Manuel López Fernández4, DVM; Javier Pérez Sanz4, DVM; Antonio López-Sebastián2, DVM, PhD; Julián Santiago-Moreno2, DVM, PhD
1Zoo-Aquarium Madrid, Madrid, Spain; 2Department of Animal Reproduction, INIA, Madrid, Spain; 3Zoológico de Córdoba, Córdoba, Spain; 4Zoológico de Guadalajara, Guadalajara, Spain


Sperm cryopreservation is the process of cooling spermatozoa to very low sub-zero temperatures for prolonged storage. A conventional freezing process requires slow cooling rates and long equilibration periods. Ultra-rapid freezing is an alternative method of cryopreservation that can be used to preserve sperm of certain species because it requires little equipment; it is quick, simple and less expensive than conventional methods of freezing.2 Cryopreservation of spermatozoa has been found to affect morphometry of the sperm head and some evidences suggest that sperm head dimensions may be an indicator of sperm cryosurvivability.1 The objective of this study was to evaluate sperm head morphometry from several wild species subjected to each methods of freezing (conventional vs ultra-rapid), and to investigate their possible relationships with freezability.

Twenty-five epididymal sperm samples of 14 different species (Iberian ibex N=3, chamois N=3, mouflon N=3, European wisent N=1, sitatunga N=1, Iberian red deer N=2, fallow deer N=3, dama gazelle N=1, dorcas gazelle N=1, giraffe N=2, brown bear N=1, dolphin N=2, koala N=1 and macaque N=1) and twenty-seven ejaculated samples of 11 different species (Aoudad N=4, Iberian ibex N=3, chamois N=3, mouflon N=3, sitatunga N=1, white-tailed gnu N=1, Iberian red deer N=2, Pere David’s deer N=1, fallow deer N=6, muntjac N=2 and giant panda N=1) were collected in different Spanish zoological gardens. The epididymal sperm were collected by the ‘flushing’ method3, while the ejaculated sperm were obtained by the TUMASG technique4. Each sample was subdivided into two aliquots; one for conventional freezing method and the other for ultra-rapid freezing2. Specific diluents were adapted to each species. For each case, the sperm parameters of the samples were evaluated before and after the cryopreservation. Sperm head morphometric analysis was performed using computer-aided sperm analysis system (CASA-morph). Briefly, samples smears were air-dried and stained using Hemacolor. Sperm head length, width, area and perimeter data were acquired for 100 cells. Both, koala’s and dolphin’s slides were subjected to computerized morphometric analysis using Motic Image Advanced V.3.0 software.

The average values of the head dimensions of epididymal samples analyzed varied from the smallest (dolphin; head length 4.418±0.122 μm, width 2.717±0.089 μm, perimeter 9.159±0.047 μm and area 7.189±0.049 μm2), to the biggest (dama gazelle; head length 9.458±0.041 μm, width 5.690±0.025 μm, perimeter 26.016±0.089 μm and area 43.746±0.291 μm2).

The average values of the head dimensions of ejaculated samples analyzed varied from the smallest (giant panda; head length 5.046±0.030 μm, width 3.741±0.023 μm perimeter 14.968±0.074 μm and area 15.926±0.150 μm2) to the biggest (Mouflon; head length 9.261±0.020 μm, width 5.023±0.008 μm perimeter 24.457±0.038 μm and area 38.258±0.104 μm2).

Ultra-rapid freezing returned better results than the conventional freezing protocol for epididymal sperm from dorcas gazelle, dama gazelle, giraffe, and brown bear. For ejaculated sperm samples, ultra-rapid freezing offered worse results than the conventional freezing protocol, although with acceptable motility (20%) and viability rates (49%) in giant panda.

Epididymal sperm head area decreased (P<0.05), in all species, after conventional freezing-thawing processes, except in both dolphin and giraffe sperm. In contrast, after ultra-rapid freezing-thawing processes, epididymal sperm head area increased (P<0.05) in giraffe, European wisent, fallow deer, Iberian red deer, dolphin and koala species. Interestingly, the sperm head area wasn’t affected by freezing-thawing process in dorcas gazelle. Average results of ejaculated samples showed that both freezing-thawing processes reduced significantly (P<0.05) the sperm head area in all species, except in both Iberian red deer and gnu species, which sperm head area dimensions increased significantly (P<0.05).

In a few words, our results suggest that the morphometric changes during freezing-thawing process affected in a species-dependent fashion, depending of sperm origin (ejaculated or epididymal). On one hand, there were samples that showed a significant reduction in sperm head dimensions after freezing-thawing process. On the other hand, there were species that showed a significant increase in sperm head dimensions after freezing-thawing process.


Supported by MINECO grant AGL2014-52081-R

Literature Cited

1.  Gravance CG, Vishwanath R, Pitt C, Garner DL, Casey PJ. Effects of cryopreservation on bull sperm head morphometry. J Androl. 1998;19:704–709.

2.  Pradiee J, Esteso MC, Lopez-Sebastián A, Toledano-Díaz A, Castaño C, Carrizosa JA, Urrutia B, Santiago- Moreno J. 2015. Successful ultrarapid cryopreservation of wild Iberian ibex (Capra pyrenaica) spermatozoa. Theriogenology. 2015;84(9):1513–22.

3.  Santiago-Moreno J, Astorga RJ, Luque I, Coloma MA, Toledano-Díaz A, Pulido-Pastor A, Gómez-Guillamon F, Salas-Vega R, López-Sebastián A. Influence of recovery method and microbial contamination on the response to freezing-thawing in ibex (Capra pyrenaica) epididymal spermatozoa. Cryobiology. 2009;59(3):357–62.

4.  Santiago-Moreno J, Castaño C, Toledano-Díaz A, Esteso MC, López-Sebastián A. Cryopreservation of aoudad (Ammotragus lervia sahariensis) sperm obtained by transrectal ultrasound-guided massage of the accessory sex glands and electroejaculation. Theriogenology. 2013;79:383–91.


Speaker Information
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Eva Martínez-Nevado, DVM
Zoo-Aquarium de Madrid
Madrid, Spain

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