Cryobiology 68 (2014) 91–95
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Cryopreservation of epididymal stallion sperm q M. Olaciregui ⇑, L. Gil, A. Montón, V. Luño, R.A. Jerez, J.I. Martí Department of Animal Pathology, Obstetric and Reproduction Area, Faculty of Veterinary Medicine, Universidad de Zaragoza, Spain
a r t i c l e
i n f o
Article history: Received 7 October 2013 Accepted 30 December 2013 Available online 8 January 2014 Keywords: Cryopreservation Epididymal sperm Stallion Cryoprotectant Cooling rate Egg yolk
a b s t r a c t Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion’s breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen–thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was tested alone or combination with pasteurized egg yolk as substitute of fresh egg yolk. In addition, the effect of the pre-freeze stabilization on sperm quality was analyzed. Heterospermic samples obtained from stallion epididymis were collected and cryopreserved in lactose–egg-yolk extender or in the same extender with varying content of cryoprotectant and content of egg yolk, stabilized and no-stabilized. Sperm motility, viability, hypoosmotic swelling test (HOST) and acrosome integrity were evaluated post-thawing. No improvement was observed on the replacement of fresh yolk by pasteurized egg yolk, whereas the results suggest that dimethylformamide is a cryoprotectant suitable for cryopreservation of equine epididymal semen, even better than glycerol. In addition, we found that the stabilization before freezing on epididymal stallion sperm, can improve sperm quality parameters. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Cryopreserved semen is an important tool in assisted reproduction, in particular, for the equine industry sperm-freezing technology has become an area of increasing interest [31]. Although the first equine pregnancy using frozen semen was reported in 1957 [4], it has been estimated that only 30–40% of stallions produce semen that is suitable for cryopreservation, and a large inter-individual variation on sperm survival during the freezing and thawing procedures has been also reported [2,18]. These facts have limited the widespread application of frozen semen by horse industry, so that the improvement of freezing protocol and extender composition is of great importance. Sudden death, catastrophic injury, castration or any other event that makes semen collection impossible may prematurely terminate a stallion’s reproductive life [25]. Fortunately, stallion sperm capable of fertilization can be harvested from the cauda epididymis [4,15] and can be used for artificial insemination of either fresh or frozen semen [22]. Besides, a recent study demonstrated that the number of spermatozoa recovered from the cauda epididymis is higher than that recovered from artificial vagina on a single
q
Statement of funding: This research was supported by DGA (G.C.I.A. 2011.A34).
⇑ Corresponding author. Fax: +34 976761612.
E-mail addresses: [email protected], [email protected] (M. Olaciregui). 0011-2240/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cryobiol.2013.12.009
collection [23]. There are limited data regarding survival and fertility of frozen–thawed epididymal stallion sperm, although the first pregnancy using frozen–thawed stallion spermatozoa was reported in a mare inseminated with epididymal spermatozoa [4]. If sperm recovery from cauda epididymis is the last chance to obtain viable spermatozoa from a stallion, to test protocols to enhance the success rates of this biotechnology in stallions is a important point of study [25]. Glycerol has been the first cryoprotectant used [28], and it has been routinely used with success in freezing extenders for semen of many domestic or wild animals [7,38], including horses [3,19]. However, glycerol causes injury to spermatozoa during cryopreservation process (review by Fahy et al. [10]), therefore its use as a cryoprotectant could be a factor involved in poor post-thaw motility and fertility rates in frozen stallion doses [3]. Glycerol toxicity may result in protein denaturation, alteration of actin interactions and induction of protein-free membrane blisters, resulting in a detrimental effect on the fertility of fresh cooled and thawed equine semen [8,24]. Glycerol toxicity is partly due to osmotic stress, because glycerol permeates the cell membrane slower than other cryoprotectants [12]. Negative effects of glycerol have encouraged investigation on alternative cryoprotectants for equine sperm with similar properties but with less toxic effects. It was suggested that the ideal cryoprotectant must have low molecular weight, great water solubility and minimal toxicity. Most of the amides had a
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lower molecular weight compared with glycerol and these cryoprotectants may induce less osmotic damage [3,19]. Research on the effects of different cryoprotectants on ejaculated sperm shed some light on the potential beneficial effects of formamides on epididymal sperm. Equine semen is generally cryopreserved using egg yolk-based freezing extender [27]. Egg yolk protects the sperm cells from the toxic effects of seminal plasma. The amount of egg yolk required in semen diluents to provide protection against seminal plasma toxins is proportional to the amount of seminal plasma in diluted semen [32]. Egg yolk also exerts a protective effect against cold shock, primarily due to its phospholipid components [36]. Jasko et al. [14] reported that 4% egg yolk in an extender was beneficial for maintenance of spermatozoa motion characteristics after cooling. However, recent arguments against the use of egg yolk have arisen, one of which is the wide variability of composition that makes it difficult to analyze the beneficial effects of a particular compound on sperm cryopreservation. Furthermore, egg yolk introduces a risk of microbial contamination, with the subsequent production of endotoxins capable of damaging the fertilizing capacity of spermatozoa [1,5,11,27]. Pasteurized egg yolk may be used as an alternative to prepare freezing extenders and reduces microbial contamination. van Wagtendonk-De Leeuw et al. [33] reported high percentages of motile sperm after thawing in bull sperm frozen in pasteurized egg yolk extender and 56-day non-return rates higher than 67%. Besides Botu-CrioÒ diluent, which contains pasteurized egg yolk, provided excellent fertility rates for stallion sperm in other studies [20,25]. Regarding freezing protocol, Cochran et al. [6] reported that exposure of lactose-egg yolk extended semen to prolonged prefreezing cooling conditions are not required. This technique speeds up the process, and is currently being used for freezing stallion semen [20,26]. However, Woolley et al. [39] reported that a slow prefreeze cooling rate could increase the cold injury resistance of the acrosomal membrane and the plasma membrane on human sperm. In addition, Watson et al. [37] reported that if stallion spermatozoa are cooled-rapidly, they undergo irreversible membrane changes, termed cold shock. Besides, Salazar et al. [30] found recently that sperm quality with slow pre-freeze cooling rate was higher than using fast pre-freeze cooling rate. Therefore, the objective of this study was to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of replacement of glycerol by dimethylformamide (DMF), totally or partially, and fresh egg yolk by pasteurized egg yolk for freezing equine epididymal sperm. In addition we analyzed stabilization before freezing as Cochran’s protocol alternative. Materials and methods Unless otherwise indicated, all chemicals were from Sigma–Aldrich Co. (Alcohobendas, Madrid, Spain). Dimethylformamide and glycerol were from Panreac Quimica S.L.U (Barcelona, Spain), Equex Paste was from Minitub and pasteurized egg yolk was from Grupo Leche Pascual (Spain). The medium used for washing and centrifugation was Citrate– EDTA, composed of 8.33 mM glucose, 88.23 mM sodium citrate, 9.93 mM disodium EDTA, 14.28 mM sodium bicarbonate. Lactose–egg-yolk extender containing 50% (v/v) of 290 mM L-lactose, 20% (v/v) of egg yolk, 25% (v/v) of Glucose–EDTA medium (322.20 mM glucose, 12.58 mM sodium citrate, 9.93 mM disodium EDTA, 14.28 mM sodium bicarbonate), 0.5% (v/v) of Equex Paste and 5%(v/v) of glycerol, was used as control freezing extender. The freezing extender for the different treatments was the same than for the control freezing extender varying the type of egg yolk and the concentration and type of cryoprotectant.
Experimental design Six replicates were conducted, using on each replicate a heterospermic mixture from three stallion’s epididymis collected at the slaughterhouse (Mercazaragoza, Zaragoza). Five experimental freezing extenders were prepared varying the cryoprotectant concentration and egg yolk type: GP (5% glycerol and pasteurized egg yolk), G/DMFF (2.5% glycerol, 2.5% dimethylformamide and fresh egg yolk), G/DMFP (2.5% glycerol, 2.5% dimethylformamide and pasteurized egg yolk), DMFF (5% dimethylformamide and egg yolk fresh), and DMFP (5% dimethylformamide and pasteurized egg yolk). As control freezing extender we used GF (5% glycerol and fresh egg yolk). In order to determine the stabilization effect in the cryopreservation procedure, samples were frozen by two different methods. In the first of them, extended semen is packaged in straws at 22 °C and immediately exposured to freezing conditions (as described by Cochran [6]). The second procedure consist of a stabilization step with a slow cooling rate, in which semen samples were cooled to 4 °C in 2 h, before packing at 4 °C. Collection, dilution and freezing of stallion epididymis spermatozoa Sperm was collected using a retrograde flushing. The cauda epididymides were dissected and cannulated with a 25 G needle connected to 10 mL syringe with 2 mL of Citrate–EDTA at 20 °C. Then manual pressure was applied by a syringe and spermatozoa were collected in a Petri dish. Semen samples were centrifuged at 1000g for 5 min at room temperature and the supernatant was discarded. Remaining semen was slowly extended to achieve a concentration of 100 106 per mL with the medium (GF, GP, G/DMFF, G/ DMFP, DMFF, DMFP, respectively). Then, semen samples were cryopreserved using two different ways described above. After being packaged, straws were placed for 10 min in liquid nitrogen vapor, 4 cm above the level of liquid nitrogen. The straws were then plunged into liquid nitrogen and stored at 196 °C. Semen quality Frozen semen samples were thawed in circulating water at 38 °C for 30 s. Sperm motility, viability, acrosome integrity and hypoosmotic swelling test (HOST) at 0 and 2 h after incubation at 38 °C (thermoresistance test) were assessed. The percentage of total motile spermatozoa and percentage of progressively motile spermatozoa was analyzed by means of a computer assisted semen analysis (CASA) system (ISAS PROISER; Valencia; Spain). Sperm viability was evaluated using eosin–nigrosin stain [9]. A semen sample was diluted 1:1 (v/v) with stain solution (5% eosin, 10% nigrosin in a citrate solution) and smeared. Live spermatozoa remained unstained. In addition, the percentage of normal acrosome was evaluated under a phase contrast microscope. Samples were fixed in buffered 8% glutaraldehyde solution and the percentage of spermatozoa with intact acrosome was determined [29]. Membrane functional integrity was further assessed using HOST [17]. The technique consisted of incubating 10 lL of semen with 90 ll of lactose hypoosmotic solution (100 mOsm) at 37 °C for 30 min. The samples were then fixed in 8% glutaraldehyde buffered solution. The proportion of sperm with swollen or coiled tails was considered as HOST-positive. Statistical analysis Analysis was performed using SPSS 17.0 for Windows. Results were expressed as mean ± SD. Data concerning to effect of
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stabilization and egg yolk source were assessed by Mann–Whitney test. The values concerning to effect of different cryoprotector were analyzed using ANOVA. When one-way ANOVA revealed a significant effect, values were compared by post-hoc test (Duncan). The interactions among treatments were analyzed using GLM procedure. The level of significance was set at P < 0.05. Results Table 1 shows the effects of pre-freezing stabilization step on epididymal semen quality. Immediately after thawing and at 2 h, all samples showed significant (p < 0.01) high percentages of total motility, progressive motility and viability when pre-freezing stabilization step was performed. Regarding percentage of integrity acrosome was significantly higher (p < 0.01) in presence of prefreezing stabilization step after thawing. However at 2 h no significant differences were observed. No effect (p > 0.01) of pre-freezing stabilization step was observed on response to HOST at post-thaw neither at 2 h. Cryoprotectant substitution effects in epididymal stallion semen quality are reported in Table 2. After thawing, a significant increase in percentage of total and progressive motile sperm was observed in the presence of DMF alone, whereas at 2 h post-thaw, the combination DMF and glycerol increased the percentages of total and progressive motility. There were also significant differences (p < 0.01) among cryoprotectors in viability rate. An increase in spermatozoa with intact plasma membrane was observed after thawing when DMF was used alone. However, no significant
differences were found after TRT. No cryoprotector effect was observed on the percentages of intact acrosome and HOST at any time. The effects of egg yolk during cryopreservation on epididymal semen quality are shown in Table 3. After thawing, samples treated with fresh egg yolk showed significant higher percentages of all semen quality parameters than pasteurized egg yolk samples. Results were similar among treatments at 2 h. Finally, we estimated statistically possible interactions among the three factors simultaneously studied and their influence on epididymal sperm quality. There were significant differences in total and progressive motility among treatment at post-thaw and at 2 h. However, there were no significant differences in viability rate, HOST response and acrosome integrity among treatment at any time (data not shown). As shown in Figs. 1 and 2, total and progressive motility percentages were significantly higher (p < 0.01) when freezing extender contained DMF compared to glycerol. Pasteurized egg yolk samples showed again worse results than fresh egg yolk, and no relevant interactions were observed. There were also, clear differences between stabilizing and no stabilizing. Spermatozoa preserved by stabilization step showed a significant improvement (p < 0.01) in terms of sperm motility. Discussion Previous studies have shown a significant reduction in motion characteristics of stallion spermatozoa when cooled rapidly over
Table 1 Effects of stabilization on sperm quality (mean ± SD).
⁄
TM
PM
Viability
Host
Intact acrosome
Post-thaw
No-stabilization Stabilization P value
23.96 ± 16.70 40.93 ± 24.91 0.000
14.45 ± 13.49 27.85 ± 21.45 0.000
59.85 ± 9.87 67.24 ± 9.46 0.000
46.72 ± 11.39 48.47 ± 9,88 0.370
37.21 ± 7.93 40.93 ± 7.79 0.003
TRT
No-stabilization Stabilization P value
3.88 ± 4.98 10.46 ± 10.51 0.000
1.72 ± 2.66 6.26 ± 7.89 0.000
42.64 ± 7.91 50.77 ± 9.52 0,000
33.48 ± 8.86 36.34 ± 9.41 0.077
30.72 ± 5.07 31.92 ± 6.68 0.283
TM, total motility; PM, progressive motility; TRT, thermoresistance test.
Table 2 Effects of different cryoprotector added to the freezing extender on sperm quality (mean ± SD).
⁄
Time
Cryoprotector
TM
PM
Viability
Host
Intact acrosome
Post-thaw
Glycerol Glycerol/DMF DMF P value
18.67 ± 12.75a 36.33 ± 24.80b 49.55 ± 20.71c 0.000
11.30 ± 11.04a 24.93 ± 22.11b 32.76 ± 18.73c 0.000
59.23 ± 9.51a 64.57 ± 10.21b 69.89 ± 8.07c 0.000
48.55 ± 12.03ab 45.23 ± 10.22a 49.78 ± 8.37c 0.053
40.04 ± 9.42a 38.25 ± 7.85a 40.27 ± 6.69a 0.327
TRT
Glycerol Glycerol/DMF DMF P value
6.00 ± 7.17a 10.17 ± 11.23b 7.64 ± 8.86ab 0.048
3.13 ± 5.00a 6.63 ± 8.35b 3.73 ± 6.05a 0.010
46.90 ± 8.11a 46.77 ± 11.58a 49.45 ± 9.14a 0.257
37.50 ± 10.29a 33.57 ± 8.01a 34.35 ± 9.06a 0.051
32.65 ± 6.43a 30.47 ± 6.45a 31.24 ± 5.24a 0.142
TM, total motility; PM, progressive motility; TRT, thermoresistance test. Different letters within the same column represent a significant difference (p < 0.05).
Table 3 Effects of different egg yolk source added to the freezing extender on sperm quality (mean ± SD).
⁄
Time
Egg yolk
TM
PM
Viability
Host
Intact acrosome
Post-thaw
Fresh Pasteurized P value
45.82 ± 21.33 23.67 ± 20.40 0.000
34.35 ± 18.22 11.73 ± 14.38 0.000
67.89 ± 8.14 61.12 ± 10.97 0.000
52.60 ± 9.22 43.27 ± 9.59 0.000
43.89 ± 7.65 35.36 ± 5.92 0.000
TRT
Fresh Pasteurized P value
14.12 ± 9.70 2.11 ± 3.43 0.000
8.51 ± 7.83 0.76 ± 1.56 0.000
51.22 ± 9.89 44.29 ± 8.37 0.000
39.47 ± 9.01 31.09 ± 7.56 0.000
33.73 ± 5.58 21.31 ± 5.86 0.000
TM, total motility; PM, progressive motility; TRT, thermoresistance test.
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Fig. 1. Effects of interactions between the different factors studied on total and progressive sperm motility at post-thaw (Mean ± SD).
Fig. 2. Effects of interactions between the different factors studied on total and progressive sperm motility at 2 h after thawing (Mean ± SD).
the temperatures of cold shock sensitivity [16,21,34]. However, Cochran et al. [6] concluded that carrying out pre-freezing stabilization step, is not necessary and they designed a freezing protocol without stabilization step of cooling semen to 5 °C over a 30-min period prior to freezing. Conversely, in the present study on equine epididymal sperm freezing, we demonstrated that sperm quality improved when a pre-freezing stabilization step was carried out. These findings are in agreement with a recent study [30] in which ejaculated sperm were more resistant to cryoinjury after slow prefreeze cooling rate compared to immediate exposure to low cryopreservation temperatures. Woolley et al. [39] also reported that application of a slow cooling rate before freezing may have increased the resistance of the outer acrosomal membrane and overlying plasma membrane to cooling and cryoinjury. Moreover, when the effect of cryoprotectant on sperm quality was analyzed, we have found that DMF is a suitable cryoprotector for equine epididymal spermatozoa freezing. Hoffmann et al. [13] reported that ejaculated sperm cryopreserved using 4% glycerol shows slightly higher post-thaw motility compared to sperm cryopreserved using 4% dimethylformamide. Conversely, we observed that the presence of DMF in the freezing extender improved motility percentages as well as preserved the rest of epididymal sperm quality parameters with acceptable values during cryopreservation. This is an important finding because sperm motility is an essential parameter to the oocyte fertilization. Result agrees to data on ejaculated sperm reported by Medeiros et al. [19], who
claimed that amides improved significantly the frozen stallion spermatozoa fertility compared to glycerol. In addition, Alvarenga et al. [3] reported that amides protected stallion sperm from cryodamage and could improve fertility. These beneficial effects may be due to the fact that relative lower viscosity and molecular weight of the amides likely favored an enhanced permeability of these compounds into the plasma membrane, resulting in a reduction osmotic damage on stallion spermatozoa. It is known that the presence of egg yolk in freezing extender is beneficial for maintaining sperm motility after cooling [14]. It is routinely included in cryopreservation protocols to minimize cold-shock effects, and it also reduces the toxic effects of seminal plasma and provides substrates to neutralize H2O2 that is produced by the sperm during metabolism [35]. All this indicate that egg yolk, or any similar product, is necessary to prepare stallion freezing extender. Papa et al. [26] concluded that an extender containing soybean lecithin as the lipid/lipoprotein source can be an alternative to conventional extenders that contain skim milk and/or egg yolk, due to it preserved sperm motility and plasma membrane integrity similar to the conventional extender containing egg yolk. In addition, Papa et al. and Melo et al. [20,25] demonstrated high percentages of fertility when they used Botu-CrioÒ, which contain pasteurized egg yolk, for cryopreserving stallion sperm. However, in the present study, sperm quality was not improved by fresh egg yolk replacement for pasteurized on epididymal stallion sperm. In fact, data showed that pasteurized egg
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yolk in the freezing extender had a negative effect on parameters studied. Differences may be to sperm (ejaculated vs. epididymal) and/or pasteurized egg yolk origin. Therefore further studies on pasteurized egg yolk are required to ensure improvement on fertility parameters and verify the beneficial effect of pasteurized egg yolk with lower variability of its composition than fresh egg yolk. Finally, after analyzing interactions effects among the three variables studied, cryoprotectants, egg yolk and stabilization step, we conclude that the combination of dimethylformamide and fresh egg yolk in freezing extender improve post- thawed epididymal sperm quality, and a pre-freezing stabilization step is necessary. Although conclusions from this study are in agreement with previous results reported for ejaculated sperm about cryoprotectant added to the freezing extender and pre-freezing stabilization step, further research is required to obtain more information about the source of pasteurized egg yolk which allows to avoid fresh egg yolk variability. Acknowledgment This study was supported by Government of Aragon Research Groups (G.C.I.A. 2011. A34). References [1] V.A. Aires, K.D. Hinsch, F. Mueller-Schoesser, K. Bogner, S. Mueller-Scloesser, E. Hinsch, In vitro and in vivo comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of bovine semen, Theriogenology 60 (2003) 269–279. [2] M.A. Alvarenga, K.M. Leao, F.O. Papa, F.C. Landim-Alvarenga, A.S.L. Medeiros, G.M. Gomes, The use of alternative cryoprotectors for freezing stallion semen, Proc. Workshop Transport. Gametes Embryos, Havemeyer Foundation 12 (2003) 74–76. [3] M.A. Alvarenga, F.O. Papa, F.C. Landim-Alvarenga, A.S.L. Medeiros, Amides as cryoprotectants for freezing stallion semen: a review, Anim. Reprod. Sci. 89 (2005) 105–113. [4] C.A. Barker, S.C.C. Gandier, Pregnancy in a mare resulting from frozen epididymal spermatozoa, Can. J. Comp. Med. Vet. Sci. 21 (2) (1957) 47–51. [5] S. Bousseau, B. Marguant-Le Guienne, B. Guerin, A. Camus, M. Lechat, Comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin-based diluents, Theriogenology 50 (1998) 699–706. [6] J.D. Cochran, R.P. Amann, D.P. Froman, B.W. Pickett, Effects of centrifugation, glycerol level, cooling to 5 °C, freezing rate and thawing rate on the post-thaw motility of equine sperm, Theriogenology 22 (1984) 25–39. [7] M.R. Curry, B.J. Redding, P.F. Watson, Determination of water permeability coefficient and its activation energy for rabbit spermatozoa, Cryobiology 32 (1995) 175–181. [8] D.S. Demick, J.L. Voss, B.W. Pickett, Effect of cooling, storage with glycerolization and spermatozoa number on equine fertility, J. Anim. Sci. 43 (1976) 633–637. [9] H.M. Dott, G.C. Foster, A technique for studying the morphology of mammalian spermatozoa which is eosinophilic in a differential ‘‘live–dead’’ stain, J. Soc. Reprod. Fertil. 29 (1972) 443. [10] G.M. Fahy, T.H. Lilley, H. Linsdell, M.S.J. Douglas, H.T. Meryman, Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms, Cryobiology 27 (1990) 247–268. [11] J. Gil, M. Rodriguez-Irazoqui, N. Lundeheim, L. Söderquist, H. RodríguezMartínez, Fertility of ram semen frozen in BioexcellÒ and used for cervical artificial insemination, Theriogenology 59 (2003) 1157–1170. [12] J.A. Gilmore, L.E. McGann, J. Liu, D.Y. Gao, A.T. Peter, F.W. Kleinhans, J.K. Critser, Effects of cryoprotectant solutes on water permeability of human spermatozoa, Biol. Reprod. 53 (1995) 985–995. [13] N. Hoffmann, H. Oldenhof, C. Morandini, K. Rohn, H. Sieme, Optimal concentrations of cryoprotective agents for semen from stallions that are classified ‘good’ or ‘poor’ for freezing, Anim. Reprod. Sci. 125 (2011) 112–118.
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[14] D.J. Jasko, J.A. Hathaway, V.L. Schaltenbrand, W.D. Simper, E.L. Squires, Effect of seminal plasma and egg yolk on motion characteristics of cooled stallion spermatozoa, Theriogenology 37 (1992) 1241–1252. [15] L. Johnson, R.P. Amann, B.W. Pickett, Maturation of equine epididymal spermatozoa, Am. J. Vet. Res. 41 (8) (1980) 1190–1196. [16] J.P. Kayser, R.P. Amann, R.K. Shideler, E.L. Squires, D.J. Jasko, B.W. Pickett, Effects of linear cooling rate on motion characteristics of stallion spermatozoa, Theriogenology 22 (1984) 291–304. [17] M.A. Lagares, Bestimmung der osmotischen Resistenz vom Hengstsamenzellen Tieräztliche, Hochschule Hannover Diss (1995) 1–120. [18] P.R. Loomis, J.K. Graham, Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols, Anim. Reprod. Sci. 105 (2008) 119–128. [19] A.S.L. Medeiros, G. Gomes, M.T. Carmo, F.O. Papa, M.A. Alvarenga, Cryopreservation of stallion sperm using different amides, Theriogenology 58 (2002) 273–276. [20] C.M. Melo, F.O. Papa, M.A. Alvarenga, J.A. Dell’Aqua Jr., F.S. Zahn, C. Orlandi, I. Martin, Influence of semen storage and cryoprotectant on post-thaw viability and fertility of stallion spermatozoa, J. Equine Vet. Sci. 27 (2007) 171–175. [21] D.M. Moran, D.J. Jasko, E.L. Squires, R.P. Amann, Determination of temperature and cooling rate which induce cold shock in stallion spermatozoa, Theriogenology 38 (1992) 601–614. [22] L. Morris, C. Tiplady, W.R. Allen, The in vivo fertility of cauda epididymal spermatozoa in the horse, Theriogenology 58 (2002) 643–646. [23] P.R. Muradás, R.R. Weiss, L.E. Kozicki, L.C. Granemann, I.W. Santos, C.T. Pimpao, Some viability parameters from equine spermatozoa harvested by artificial vagina and by epididymal tail washing, J. Vet. Sci. 11 (2006) 69–74. [24] M.M. Pace, J.J. Sullivan, Effect of insemination, number of spermatozoa and extender components on the pregnancy rate in mares inseminated with frozen stallion semen, J. Soc. Reprod. Fertil. 23 (1975) 115–121. [25] F.O. Papa, C.M. Melo, E.G. Fioratti, J.A. Dell’Aqua Jr., F.S. Zahn, M.A. Alvarenga, Freezing of stallion epididymal sperm, Anim. Reprod. Sci. 107 (2008) 293–301. [26] F.O. Papa, G. Barcelos-Felício, C. Marini-Melo-Ona, M.A. Alvarenga, C. De Vita Trinque, J.N.P. Puoli-Filho, J.A. Dell’Aqua Jr., Replacing egg yolk with soybean lecithin in the cryopreservation of stallion semen, Anim. Reprod. Sci. 129 (2011) 73–77. [27] E. Pillet, G. Duchamp, F. Batellier, V. Beaumal, M. Anton, S. Desherces, E. Schmitt, M. Magistrini, Egg yolk plasma can replace egg yolk in stallion freezing extenders, Theriogenology 75 (2001) 105–114. [28] C. Polge, A.U. Smith, A.S. Parks, Revival of spermatozoa after vitrification and dehydration at low temperatures, Nature 164 (1949) 666. [29] V.G. Pursel, L.A. Johnson, Glutaraldehyde fixation of boar spermatozoa for acrosome evaluation, Theriogenology 1 (1974) 63. [30] J.L. Salazar, S. Teague, C. Love, S. Brinsko, T. Blanchard, D. Varner, Effect of cryopreservation protocol on post-thaw characteristics of stallion sperm, Theriogenology 76 (2011) 409–418. 31 J.C. Samper, A.J. Estrada, A.O. McKinnon, Insemination with Frozen Semen, in: J.C. Samper, J.F. Pycock, A.O. MacKinnon (Eds.), Current Therapy in Equine Reproduction Saunders, Elsevier, St Luois, Missouri, 2007, pp. 285–289. [32] P. Shannon, B. Curson, The effect of egg yolk levels on bovine seminal plasma toxins, in: Proc. 7th Int. Congress Animal Reproduction Artificial Insemination, Munich, Germany 4 (1972) 1363–1367. [33] A.M. van Wagtendonk-De Leeuw, R.M. Haring, L.M.T.E. Kaal-Lansbergen, J.H.G. den Daas, Fertility results using bovine semen cryopreserved with extenders based on egg yolk and soybean extract, Theriogenology 54 (2000) 57–67. [34] D.D. Vamer, T.L. Blanchard, C.L. Love, M.C. Garcia, R.M. Kenney, Effects of cooling rate and storage temperature on equine spermatozoa motility parameters, Theriogenology 29 (1988) 1044–1054. [35] R. Vishwanath, P. Shannon, Storage of bovine semen in liquid and frozen state, Anim. Reprod. Sci. 62 (2000) 23–53. [36] P.F. Watson, The roles of lipid and protein in the protection of ram spermatozoa at 5% by egg-yolk lipoprotein, J. Soc. Reprod. Fertil. 62 (1981) 483–492. [37] P.F. Watson, J.M. Plummer, W.E. Allen, Quantitative assessment of membrane damage in cold-shocked spermatozoa of stallions, J. Soc. Reprod. Fertil. 35 (1987) 651–653. [38] P.F. Watson, Recent development and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function, Reprod. Fertil. Dev. 7 (4) (1995) 871–891. [39] D.M. Woolley, D.W. Richardson, Ultrastructural injury to human spermatozoa after freezing and thawing, J. Soc. Reprod. Fertil. 53 (1978) 389–394.
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Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Cryopreservation of epididymal stallion sperm q M. Olaciregui ⇑, L. Gil, A. Montón, V. Luño, R.A. Jerez, J.I. Martí Department of Animal Pathology, Obstetric and Reproduction Area, Faculty of Veterinary Medicine, Universidad de Zaragoza, Spain
a r t i c l e
i n f o
Article history: Received 7 October 2013 Accepted 30 December 2013 Available online 8 January 2014 Keywords: Cryopreservation Epididymal sperm Stallion Cryoprotectant Cooling rate Egg yolk
a b s t r a c t Any event that makes semen collection or mating impossible, such as death, castration, or injury, may terminate a stallion’s breeding career. Fortunately, stallion sperm which are capable of fertilization can be harvested from the epididymis, and frozen for future use. However, the fertility of frozen–thawed epididymal sperm has been found to be lower than that of ejaculated sperm. Therefore, this study aimed to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of different cryoprotectants and freezing protocols on sperm quality. Dimethylformamide was tested alone or combination with pasteurized egg yolk as substitute of fresh egg yolk. In addition, the effect of the pre-freeze stabilization on sperm quality was analyzed. Heterospermic samples obtained from stallion epididymis were collected and cryopreserved in lactose–egg-yolk extender or in the same extender with varying content of cryoprotectant and content of egg yolk, stabilized and no-stabilized. Sperm motility, viability, hypoosmotic swelling test (HOST) and acrosome integrity were evaluated post-thawing. No improvement was observed on the replacement of fresh yolk by pasteurized egg yolk, whereas the results suggest that dimethylformamide is a cryoprotectant suitable for cryopreservation of equine epididymal semen, even better than glycerol. In addition, we found that the stabilization before freezing on epididymal stallion sperm, can improve sperm quality parameters. Ó 2014 Elsevier Inc. All rights reserved.
Introduction Cryopreserved semen is an important tool in assisted reproduction, in particular, for the equine industry sperm-freezing technology has become an area of increasing interest [31]. Although the first equine pregnancy using frozen semen was reported in 1957 [4], it has been estimated that only 30–40% of stallions produce semen that is suitable for cryopreservation, and a large inter-individual variation on sperm survival during the freezing and thawing procedures has been also reported [2,18]. These facts have limited the widespread application of frozen semen by horse industry, so that the improvement of freezing protocol and extender composition is of great importance. Sudden death, catastrophic injury, castration or any other event that makes semen collection impossible may prematurely terminate a stallion’s reproductive life [25]. Fortunately, stallion sperm capable of fertilization can be harvested from the cauda epididymis [4,15] and can be used for artificial insemination of either fresh or frozen semen [22]. Besides, a recent study demonstrated that the number of spermatozoa recovered from the cauda epididymis is higher than that recovered from artificial vagina on a single
q
Statement of funding: This research was supported by DGA (G.C.I.A. 2011.A34).
⇑ Corresponding author. Fax: +34 976761612.
E-mail addresses: [email protected], [email protected] (M. Olaciregui). 0011-2240/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cryobiol.2013.12.009
collection [23]. There are limited data regarding survival and fertility of frozen–thawed epididymal stallion sperm, although the first pregnancy using frozen–thawed stallion spermatozoa was reported in a mare inseminated with epididymal spermatozoa [4]. If sperm recovery from cauda epididymis is the last chance to obtain viable spermatozoa from a stallion, to test protocols to enhance the success rates of this biotechnology in stallions is a important point of study [25]. Glycerol has been the first cryoprotectant used [28], and it has been routinely used with success in freezing extenders for semen of many domestic or wild animals [7,38], including horses [3,19]. However, glycerol causes injury to spermatozoa during cryopreservation process (review by Fahy et al. [10]), therefore its use as a cryoprotectant could be a factor involved in poor post-thaw motility and fertility rates in frozen stallion doses [3]. Glycerol toxicity may result in protein denaturation, alteration of actin interactions and induction of protein-free membrane blisters, resulting in a detrimental effect on the fertility of fresh cooled and thawed equine semen [8,24]. Glycerol toxicity is partly due to osmotic stress, because glycerol permeates the cell membrane slower than other cryoprotectants [12]. Negative effects of glycerol have encouraged investigation on alternative cryoprotectants for equine sperm with similar properties but with less toxic effects. It was suggested that the ideal cryoprotectant must have low molecular weight, great water solubility and minimal toxicity. Most of the amides had a
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lower molecular weight compared with glycerol and these cryoprotectants may induce less osmotic damage [3,19]. Research on the effects of different cryoprotectants on ejaculated sperm shed some light on the potential beneficial effects of formamides on epididymal sperm. Equine semen is generally cryopreserved using egg yolk-based freezing extender [27]. Egg yolk protects the sperm cells from the toxic effects of seminal plasma. The amount of egg yolk required in semen diluents to provide protection against seminal plasma toxins is proportional to the amount of seminal plasma in diluted semen [32]. Egg yolk also exerts a protective effect against cold shock, primarily due to its phospholipid components [36]. Jasko et al. [14] reported that 4% egg yolk in an extender was beneficial for maintenance of spermatozoa motion characteristics after cooling. However, recent arguments against the use of egg yolk have arisen, one of which is the wide variability of composition that makes it difficult to analyze the beneficial effects of a particular compound on sperm cryopreservation. Furthermore, egg yolk introduces a risk of microbial contamination, with the subsequent production of endotoxins capable of damaging the fertilizing capacity of spermatozoa [1,5,11,27]. Pasteurized egg yolk may be used as an alternative to prepare freezing extenders and reduces microbial contamination. van Wagtendonk-De Leeuw et al. [33] reported high percentages of motile sperm after thawing in bull sperm frozen in pasteurized egg yolk extender and 56-day non-return rates higher than 67%. Besides Botu-CrioÒ diluent, which contains pasteurized egg yolk, provided excellent fertility rates for stallion sperm in other studies [20,25]. Regarding freezing protocol, Cochran et al. [6] reported that exposure of lactose-egg yolk extended semen to prolonged prefreezing cooling conditions are not required. This technique speeds up the process, and is currently being used for freezing stallion semen [20,26]. However, Woolley et al. [39] reported that a slow prefreeze cooling rate could increase the cold injury resistance of the acrosomal membrane and the plasma membrane on human sperm. In addition, Watson et al. [37] reported that if stallion spermatozoa are cooled-rapidly, they undergo irreversible membrane changes, termed cold shock. Besides, Salazar et al. [30] found recently that sperm quality with slow pre-freeze cooling rate was higher than using fast pre-freeze cooling rate. Therefore, the objective of this study was to optimize the fertility of frozen epididymal stallion sperm by investigating the effects of replacement of glycerol by dimethylformamide (DMF), totally or partially, and fresh egg yolk by pasteurized egg yolk for freezing equine epididymal sperm. In addition we analyzed stabilization before freezing as Cochran’s protocol alternative. Materials and methods Unless otherwise indicated, all chemicals were from Sigma–Aldrich Co. (Alcohobendas, Madrid, Spain). Dimethylformamide and glycerol were from Panreac Quimica S.L.U (Barcelona, Spain), Equex Paste was from Minitub and pasteurized egg yolk was from Grupo Leche Pascual (Spain). The medium used for washing and centrifugation was Citrate– EDTA, composed of 8.33 mM glucose, 88.23 mM sodium citrate, 9.93 mM disodium EDTA, 14.28 mM sodium bicarbonate. Lactose–egg-yolk extender containing 50% (v/v) of 290 mM L-lactose, 20% (v/v) of egg yolk, 25% (v/v) of Glucose–EDTA medium (322.20 mM glucose, 12.58 mM sodium citrate, 9.93 mM disodium EDTA, 14.28 mM sodium bicarbonate), 0.5% (v/v) of Equex Paste and 5%(v/v) of glycerol, was used as control freezing extender. The freezing extender for the different treatments was the same than for the control freezing extender varying the type of egg yolk and the concentration and type of cryoprotectant.
Experimental design Six replicates were conducted, using on each replicate a heterospermic mixture from three stallion’s epididymis collected at the slaughterhouse (Mercazaragoza, Zaragoza). Five experimental freezing extenders were prepared varying the cryoprotectant concentration and egg yolk type: GP (5% glycerol and pasteurized egg yolk), G/DMFF (2.5% glycerol, 2.5% dimethylformamide and fresh egg yolk), G/DMFP (2.5% glycerol, 2.5% dimethylformamide and pasteurized egg yolk), DMFF (5% dimethylformamide and egg yolk fresh), and DMFP (5% dimethylformamide and pasteurized egg yolk). As control freezing extender we used GF (5% glycerol and fresh egg yolk). In order to determine the stabilization effect in the cryopreservation procedure, samples were frozen by two different methods. In the first of them, extended semen is packaged in straws at 22 °C and immediately exposured to freezing conditions (as described by Cochran [6]). The second procedure consist of a stabilization step with a slow cooling rate, in which semen samples were cooled to 4 °C in 2 h, before packing at 4 °C. Collection, dilution and freezing of stallion epididymis spermatozoa Sperm was collected using a retrograde flushing. The cauda epididymides were dissected and cannulated with a 25 G needle connected to 10 mL syringe with 2 mL of Citrate–EDTA at 20 °C. Then manual pressure was applied by a syringe and spermatozoa were collected in a Petri dish. Semen samples were centrifuged at 1000g for 5 min at room temperature and the supernatant was discarded. Remaining semen was slowly extended to achieve a concentration of 100 106 per mL with the medium (GF, GP, G/DMFF, G/ DMFP, DMFF, DMFP, respectively). Then, semen samples were cryopreserved using two different ways described above. After being packaged, straws were placed for 10 min in liquid nitrogen vapor, 4 cm above the level of liquid nitrogen. The straws were then plunged into liquid nitrogen and stored at 196 °C. Semen quality Frozen semen samples were thawed in circulating water at 38 °C for 30 s. Sperm motility, viability, acrosome integrity and hypoosmotic swelling test (HOST) at 0 and 2 h after incubation at 38 °C (thermoresistance test) were assessed. The percentage of total motile spermatozoa and percentage of progressively motile spermatozoa was analyzed by means of a computer assisted semen analysis (CASA) system (ISAS PROISER; Valencia; Spain). Sperm viability was evaluated using eosin–nigrosin stain [9]. A semen sample was diluted 1:1 (v/v) with stain solution (5% eosin, 10% nigrosin in a citrate solution) and smeared. Live spermatozoa remained unstained. In addition, the percentage of normal acrosome was evaluated under a phase contrast microscope. Samples were fixed in buffered 8% glutaraldehyde solution and the percentage of spermatozoa with intact acrosome was determined [29]. Membrane functional integrity was further assessed using HOST [17]. The technique consisted of incubating 10 lL of semen with 90 ll of lactose hypoosmotic solution (100 mOsm) at 37 °C for 30 min. The samples were then fixed in 8% glutaraldehyde buffered solution. The proportion of sperm with swollen or coiled tails was considered as HOST-positive. Statistical analysis Analysis was performed using SPSS 17.0 for Windows. Results were expressed as mean ± SD. Data concerning to effect of
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stabilization and egg yolk source were assessed by Mann–Whitney test. The values concerning to effect of different cryoprotector were analyzed using ANOVA. When one-way ANOVA revealed a significant effect, values were compared by post-hoc test (Duncan). The interactions among treatments were analyzed using GLM procedure. The level of significance was set at P < 0.05. Results Table 1 shows the effects of pre-freezing stabilization step on epididymal semen quality. Immediately after thawing and at 2 h, all samples showed significant (p < 0.01) high percentages of total motility, progressive motility and viability when pre-freezing stabilization step was performed. Regarding percentage of integrity acrosome was significantly higher (p < 0.01) in presence of prefreezing stabilization step after thawing. However at 2 h no significant differences were observed. No effect (p > 0.01) of pre-freezing stabilization step was observed on response to HOST at post-thaw neither at 2 h. Cryoprotectant substitution effects in epididymal stallion semen quality are reported in Table 2. After thawing, a significant increase in percentage of total and progressive motile sperm was observed in the presence of DMF alone, whereas at 2 h post-thaw, the combination DMF and glycerol increased the percentages of total and progressive motility. There were also significant differences (p < 0.01) among cryoprotectors in viability rate. An increase in spermatozoa with intact plasma membrane was observed after thawing when DMF was used alone. However, no significant
differences were found after TRT. No cryoprotector effect was observed on the percentages of intact acrosome and HOST at any time. The effects of egg yolk during cryopreservation on epididymal semen quality are shown in Table 3. After thawing, samples treated with fresh egg yolk showed significant higher percentages of all semen quality parameters than pasteurized egg yolk samples. Results were similar among treatments at 2 h. Finally, we estimated statistically possible interactions among the three factors simultaneously studied and their influence on epididymal sperm quality. There were significant differences in total and progressive motility among treatment at post-thaw and at 2 h. However, there were no significant differences in viability rate, HOST response and acrosome integrity among treatment at any time (data not shown). As shown in Figs. 1 and 2, total and progressive motility percentages were significantly higher (p < 0.01) when freezing extender contained DMF compared to glycerol. Pasteurized egg yolk samples showed again worse results than fresh egg yolk, and no relevant interactions were observed. There were also, clear differences between stabilizing and no stabilizing. Spermatozoa preserved by stabilization step showed a significant improvement (p < 0.01) in terms of sperm motility. Discussion Previous studies have shown a significant reduction in motion characteristics of stallion spermatozoa when cooled rapidly over
Table 1 Effects of stabilization on sperm quality (mean ± SD).
⁄
TM
PM
Viability
Host
Intact acrosome
Post-thaw
No-stabilization Stabilization P value
23.96 ± 16.70 40.93 ± 24.91 0.000
14.45 ± 13.49 27.85 ± 21.45 0.000
59.85 ± 9.87 67.24 ± 9.46 0.000
46.72 ± 11.39 48.47 ± 9,88 0.370
37.21 ± 7.93 40.93 ± 7.79 0.003
TRT
No-stabilization Stabilization P value
3.88 ± 4.98 10.46 ± 10.51 0.000
1.72 ± 2.66 6.26 ± 7.89 0.000
42.64 ± 7.91 50.77 ± 9.52 0,000
33.48 ± 8.86 36.34 ± 9.41 0.077
30.72 ± 5.07 31.92 ± 6.68 0.283
TM, total motility; PM, progressive motility; TRT, thermoresistance test.
Table 2 Effects of different cryoprotector added to the freezing extender on sperm quality (mean ± SD).
⁄
Time
Cryoprotector
TM
PM
Viability
Host
Intact acrosome
Post-thaw
Glycerol Glycerol/DMF DMF P value
18.67 ± 12.75a 36.33 ± 24.80b 49.55 ± 20.71c 0.000
11.30 ± 11.04a 24.93 ± 22.11b 32.76 ± 18.73c 0.000
59.23 ± 9.51a 64.57 ± 10.21b 69.89 ± 8.07c 0.000
48.55 ± 12.03ab 45.23 ± 10.22a 49.78 ± 8.37c 0.053
40.04 ± 9.42a 38.25 ± 7.85a 40.27 ± 6.69a 0.327
TRT
Glycerol Glycerol/DMF DMF P value
6.00 ± 7.17a 10.17 ± 11.23b 7.64 ± 8.86ab 0.048
3.13 ± 5.00a 6.63 ± 8.35b 3.73 ± 6.05a 0.010
46.90 ± 8.11a 46.77 ± 11.58a 49.45 ± 9.14a 0.257
37.50 ± 10.29a 33.57 ± 8.01a 34.35 ± 9.06a 0.051
32.65 ± 6.43a 30.47 ± 6.45a 31.24 ± 5.24a 0.142
TM, total motility; PM, progressive motility; TRT, thermoresistance test. Different letters within the same column represent a significant difference (p < 0.05).
Table 3 Effects of different egg yolk source added to the freezing extender on sperm quality (mean ± SD).
⁄
Time
Egg yolk
TM
PM
Viability
Host
Intact acrosome
Post-thaw
Fresh Pasteurized P value
45.82 ± 21.33 23.67 ± 20.40 0.000
34.35 ± 18.22 11.73 ± 14.38 0.000
67.89 ± 8.14 61.12 ± 10.97 0.000
52.60 ± 9.22 43.27 ± 9.59 0.000
43.89 ± 7.65 35.36 ± 5.92 0.000
TRT
Fresh Pasteurized P value
14.12 ± 9.70 2.11 ± 3.43 0.000
8.51 ± 7.83 0.76 ± 1.56 0.000
51.22 ± 9.89 44.29 ± 8.37 0.000
39.47 ± 9.01 31.09 ± 7.56 0.000
33.73 ± 5.58 21.31 ± 5.86 0.000
TM, total motility; PM, progressive motility; TRT, thermoresistance test.
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Fig. 1. Effects of interactions between the different factors studied on total and progressive sperm motility at post-thaw (Mean ± SD).
Fig. 2. Effects of interactions between the different factors studied on total and progressive sperm motility at 2 h after thawing (Mean ± SD).
the temperatures of cold shock sensitivity [16,21,34]. However, Cochran et al. [6] concluded that carrying out pre-freezing stabilization step, is not necessary and they designed a freezing protocol without stabilization step of cooling semen to 5 °C over a 30-min period prior to freezing. Conversely, in the present study on equine epididymal sperm freezing, we demonstrated that sperm quality improved when a pre-freezing stabilization step was carried out. These findings are in agreement with a recent study [30] in which ejaculated sperm were more resistant to cryoinjury after slow prefreeze cooling rate compared to immediate exposure to low cryopreservation temperatures. Woolley et al. [39] also reported that application of a slow cooling rate before freezing may have increased the resistance of the outer acrosomal membrane and overlying plasma membrane to cooling and cryoinjury. Moreover, when the effect of cryoprotectant on sperm quality was analyzed, we have found that DMF is a suitable cryoprotector for equine epididymal spermatozoa freezing. Hoffmann et al. [13] reported that ejaculated sperm cryopreserved using 4% glycerol shows slightly higher post-thaw motility compared to sperm cryopreserved using 4% dimethylformamide. Conversely, we observed that the presence of DMF in the freezing extender improved motility percentages as well as preserved the rest of epididymal sperm quality parameters with acceptable values during cryopreservation. This is an important finding because sperm motility is an essential parameter to the oocyte fertilization. Result agrees to data on ejaculated sperm reported by Medeiros et al. [19], who
claimed that amides improved significantly the frozen stallion spermatozoa fertility compared to glycerol. In addition, Alvarenga et al. [3] reported that amides protected stallion sperm from cryodamage and could improve fertility. These beneficial effects may be due to the fact that relative lower viscosity and molecular weight of the amides likely favored an enhanced permeability of these compounds into the plasma membrane, resulting in a reduction osmotic damage on stallion spermatozoa. It is known that the presence of egg yolk in freezing extender is beneficial for maintaining sperm motility after cooling [14]. It is routinely included in cryopreservation protocols to minimize cold-shock effects, and it also reduces the toxic effects of seminal plasma and provides substrates to neutralize H2O2 that is produced by the sperm during metabolism [35]. All this indicate that egg yolk, or any similar product, is necessary to prepare stallion freezing extender. Papa et al. [26] concluded that an extender containing soybean lecithin as the lipid/lipoprotein source can be an alternative to conventional extenders that contain skim milk and/or egg yolk, due to it preserved sperm motility and plasma membrane integrity similar to the conventional extender containing egg yolk. In addition, Papa et al. and Melo et al. [20,25] demonstrated high percentages of fertility when they used Botu-CrioÒ, which contain pasteurized egg yolk, for cryopreserving stallion sperm. However, in the present study, sperm quality was not improved by fresh egg yolk replacement for pasteurized on epididymal stallion sperm. In fact, data showed that pasteurized egg
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yolk in the freezing extender had a negative effect on parameters studied. Differences may be to sperm (ejaculated vs. epididymal) and/or pasteurized egg yolk origin. Therefore further studies on pasteurized egg yolk are required to ensure improvement on fertility parameters and verify the beneficial effect of pasteurized egg yolk with lower variability of its composition than fresh egg yolk. Finally, after analyzing interactions effects among the three variables studied, cryoprotectants, egg yolk and stabilization step, we conclude that the combination of dimethylformamide and fresh egg yolk in freezing extender improve post- thawed epididymal sperm quality, and a pre-freezing stabilization step is necessary. Although conclusions from this study are in agreement with previous results reported for ejaculated sperm about cryoprotectant added to the freezing extender and pre-freezing stabilization step, further research is required to obtain more information about the source of pasteurized egg yolk which allows to avoid fresh egg yolk variability. Acknowledgment This study was supported by Government of Aragon Research Groups (G.C.I.A. 2011. A34). References [1] V.A. Aires, K.D. Hinsch, F. Mueller-Schoesser, K. Bogner, S. Mueller-Scloesser, E. Hinsch, In vitro and in vivo comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of bovine semen, Theriogenology 60 (2003) 269–279. [2] M.A. Alvarenga, K.M. Leao, F.O. Papa, F.C. Landim-Alvarenga, A.S.L. Medeiros, G.M. Gomes, The use of alternative cryoprotectors for freezing stallion semen, Proc. Workshop Transport. Gametes Embryos, Havemeyer Foundation 12 (2003) 74–76. [3] M.A. Alvarenga, F.O. Papa, F.C. Landim-Alvarenga, A.S.L. Medeiros, Amides as cryoprotectants for freezing stallion semen: a review, Anim. Reprod. Sci. 89 (2005) 105–113. [4] C.A. Barker, S.C.C. Gandier, Pregnancy in a mare resulting from frozen epididymal spermatozoa, Can. J. Comp. Med. Vet. Sci. 21 (2) (1957) 47–51. [5] S. Bousseau, B. Marguant-Le Guienne, B. Guerin, A. Camus, M. Lechat, Comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin-based diluents, Theriogenology 50 (1998) 699–706. [6] J.D. Cochran, R.P. Amann, D.P. Froman, B.W. Pickett, Effects of centrifugation, glycerol level, cooling to 5 °C, freezing rate and thawing rate on the post-thaw motility of equine sperm, Theriogenology 22 (1984) 25–39. [7] M.R. Curry, B.J. Redding, P.F. Watson, Determination of water permeability coefficient and its activation energy for rabbit spermatozoa, Cryobiology 32 (1995) 175–181. [8] D.S. Demick, J.L. Voss, B.W. Pickett, Effect of cooling, storage with glycerolization and spermatozoa number on equine fertility, J. Anim. Sci. 43 (1976) 633–637. [9] H.M. Dott, G.C. Foster, A technique for studying the morphology of mammalian spermatozoa which is eosinophilic in a differential ‘‘live–dead’’ stain, J. Soc. Reprod. Fertil. 29 (1972) 443. [10] G.M. Fahy, T.H. Lilley, H. Linsdell, M.S.J. Douglas, H.T. Meryman, Cryoprotectant toxicity and cryoprotectant toxicity reduction: in search of molecular mechanisms, Cryobiology 27 (1990) 247–268. [11] J. Gil, M. Rodriguez-Irazoqui, N. Lundeheim, L. Söderquist, H. RodríguezMartínez, Fertility of ram semen frozen in BioexcellÒ and used for cervical artificial insemination, Theriogenology 59 (2003) 1157–1170. [12] J.A. Gilmore, L.E. McGann, J. Liu, D.Y. Gao, A.T. Peter, F.W. Kleinhans, J.K. Critser, Effects of cryoprotectant solutes on water permeability of human spermatozoa, Biol. Reprod. 53 (1995) 985–995. [13] N. Hoffmann, H. Oldenhof, C. Morandini, K. Rohn, H. Sieme, Optimal concentrations of cryoprotective agents for semen from stallions that are classified ‘good’ or ‘poor’ for freezing, Anim. Reprod. Sci. 125 (2011) 112–118.
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