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Benefits of TEMPOL on ram semen motility and in vitro fertility: a preliminary study
Theriogenology 63 (2005) 2243–2253 www.journals.elsevierhealth.com/periodicals/the
Benefits of TEMPOL on ram semen motility and in vitro fertility: a preliminary study Laura Maraa,*, Carla Accardoa, Susanna Pilichia, Maria Dattenaa, Fabrizio Chessaa, Bernardo Chessab, Andrea Brancaa, Pietro Cappaia a Istituto Zootecnico e Caseario per la Sardegna, 07040 Olmedo (Sassari), Italy Istituto di Patologia Speciale e Clinica Medica Veterinaria, Facolta` di Medicina Veterinaria, via Vienna, 07100 Sassari, Italy
b
Received 7 July 2004; received in revised form 28 September 2004; accepted 7 October 2004
Abstract Extending the preservation time of fresh semen is an important goal in artificial insemination programs particularly for ewes in natural oestrus, where insemination periods are longer than for ewes synchronized with hormonal treatments. The aim of this study was to evaluate the effect of the antioxidant TEMPOL (4-hydroxy-2,2,6,6tetramethylpiperidine-1-oxyl) on the maintenance in long term storage of ram semen motility and fertility. Semen from Sarda breed rams was diluted in two extenders: sodium citrate buffer with TEMPOL and skimmed milk, used as control. Samples diluted with TEMPOL were cooled at either 15 8C or 22 8C, while those diluted with skimmed milk were cooled at 15 8C. Each sample was divided into four stocks, and stored for different times (5 min, 24, 48 and 72 h). Three aliquots were taken from each stock for every storage period. One was immediately evaluated under microscope; one was used for in vitro fertilization; one was incubated for 2 h in controlled humidified atmosphere (5% CO2, 7% O2 and 88% N2) at 39 8C, then evaluated for motility and utilized for in vitro fertilization. Ram semen diluted with media containing TEMPOL demonstrated increased motility, fertility and an improved protective effect when it was stored at 15 8C. # 2004 Elsevier Inc. All rights reserved. Keywords: Semen storage; Antioxidant; Motility; Fertility; In vitro fertilization * Corresponding author. Tel.: +39 079 387 268; fax: +39 079 389 450. E-mail address: [email protected] (L. Mara). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.10.005
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1. Introduction Sheep reproduction is highly seasonal. Hormones are currently used to extend the sexual activity of females outside the natural lambing season, although consumers are beginning to react negatively to their use. Several projects are trying to develop alternatives to hormone therapy in the field of small ruminant reproduction. However, if hormones are not used to synchronize females, ovulation will take place over a longer period, and the delay between sperm collection and fertilization will be more variable. Using stored frozen or liquid semen is one of several strategies that may be used to overcome this problem. However, the use of frozen-thawed semen is limited by its relatively short-term fertility and requires employing the laborious intrauterine insemination technique, which is not easy to perform under field conditions. The practical and economic advantages of using liquid chilled semen are clear. It is cheap, easy to handle and can be used in cervical insemination. For the last ten years fresh ram semen diluted with skimmed milk and stored at 15 8C has been the most widely used method in AI programs in the Sarda breed. The fertility rate is about 50% [1]. However this semen needs to be used within 8–10 h after dilution to guarantee good fertility [2,3]. In recent years alternative methods have been suggested to improve preservation of semen quality during storage [4–7]. Recently, Foote et al. [8] found that adding antioxidants is beneficial for sperm stored for long periods. Sperm, unlike other types of cells, have a low content of substances [8,9] to protect them against the destructive effect of reactive oxygen species (ROS) [10]. Moreover, the unsaturated fatty acids content in their membranes is high. This makes them more susceptible to peroxidative damage [11]. During manipulation before storage, sperm is exposed to oxygen and visible light radiation and this may lead to ROS formation and resulting damage to sperm cell motility and genomic integrity [12–17]. In these conditions antioxidants could help to maintain sperm motility and survival [18–26]. Recent studies show that TEMPOL, a stable membrane-permeable nitroxide with SODlike activity [27], can increase in vitro sperm survival in bulls [8,28] and turkeys [29]. The aim of this study was to investigate the benefits for ram semen motility of this antioxidant under long-term storage conditions and at different temperatures. The effect of TEMPOL on semen used for in vitro fertilization of sheep oocytes is also reported.
2. Materials and methods Except where otherwise indicated, all chemicals were obtained from the Sigma (St. Louis, MO, USA). 2.1. Collection, dilution and assessment of semen Ten Sarda breed adult rams (3–6 years old), of proven high fertility were used in the experiment. Each ram was used on alternate days. Semen was collected once a day between 8:00 and 10:00 a.m. by artificial vagina. Two ejaculates were collected per session and immediately placed in a water-bath at 30 8C. The percentage of motile sperm was
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estimated under phase-contrast microscopy at 38 8C on a hot plate, while the optical density of the sperm concentration was evaluated using a calibrated spectrophotometer. Only ejaculates with an acceptable motility score higher than 3 (on a scale of 0–5, where a score of 5 indicates rapid swirling motion and 0 denotes absence of motion) and with a spermatozoa concentration higher than 3 109 ml 1 were used. Ejaculates were diluted at a concentration of 1.6 109 spermatozoa/ml at 30 8C, employing two different extenders: sodium citrate buffer (0.1 M + 5 mM MgSO4 + 2 mM fructose) [27] added with 2 mM TEMPOL (4-hydroxy 2,2,6,6-tetramethylpiperidine) [25] or skimmed milk (90 ml sterile H2O mQ + 300 mg sulphanilamide + 10 g skim milk powderOxoid (UK) + 100 mg streptomycinsulfat + 100.000 UI penicillin G) [2] used as a control. Skimmed milk is the medium routinely used in our laboratory to dilute fresh ram semen. Five minutes after dilution, semen motility was evaluated under a phase contrast microscope by placing a 10 ml drop of diluted semen on a slide covered with a glass coverslip (22 mm 22 mm). Motility evaluation was performed by focusing the binocular phase-contrast microscope at the centre of the coverslip at 250 magnification. Slide temperature was maintained at 38 8C by using a heating stage fitted to the microscope [30]. The motility rate was determined by estimating the proportion of motile and non-motile cells, while the motility score was based on the individual spermatozoa movements, using an arbitrary scale ranging from 0 to 5, as described by Evans and Maxwell [31]. Samples diluted with TEMPOL were cooled to either 15 8C (T15) or 22 8C (T22), while all those diluted with skimmed milk were cooled at 15 8C (M15). Cooling was performed as follows: to reach 15 8C, diluted semen was placed in a beaker containing water at 30 8C and a glass ampoule of glacial acetic acid. This in turn was placed in a cold storage at 15 8C. Samples cooled to 22 8C were simply left at room temperature (22 8C). Each sample was then divided in four stocks, stored for 5 min, 24, 48 and 72 h, respectively. Three aliquots of 250 ml each were taken from each stock: one was resuspended in 2 ml of its storage liquid and evaluated under microscope; another aliquot was used directly for IVF; the last one was incubated for 2 h at the following incubation conditions: 7% O2, 5% CO2 and 88% N2 at 39 8C in humidified air. Sperm motility was evaluated and the sample was then used for IVF. 2.2. Oocytes collection and maturation Sheep ovaries were obtained from a slaughterhouse and within 2–3 h were transported to the laboratory in sterile physiological saline solution at approximately 35 8C. Oocytes were collected by aspiration of the follicles (2–6 mm in diameter) with a 5 ml syringe fitted with a 20-gauge needle and placed in tissue culture medium (TCM-199) supplemented with 5% calf serum, 0.05 mg/ml heparin and 0.05 mg/ml gentamicin sulphate. Oocytes were washed in Hepes-buffered TCM 199 (H-TCM 199) supplemented with 0.4% BSA (bovine serum albumin fraction V, A 9418), and 2 mM glutamine. The medium used for maturation was bicarbonate-buffered TCM 199 with the osmolarity adjusted to 275 mOsm and containing 2 mM glutamine, 5 mg/ml follicle stimulating hormone (FSH) (Ovagen ICP, Auckland, NZ), 5 mg/ml luteinizing hormone (LH), 1 mg/ml estradiol, 0.3 mM sodium pyruvate and 100 mM cysteamine [32]. The oocytes were incubated in
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400 ml of medium in 4-well dishes (Nunc, Nunclon, Denmark) with each well containing 20–30 oocytes covered with mineral oil. All dishes were incubated at 39 8C in air in a humidified atmosphere of 5% CO2 for 24 h [33,34]. 2.3. In vitro fertilization (IVF) Following maturation, oocytes were partially stripped of surrounding cumulus cells using a fine pipette and 300 UI/ml of hyaluronidase in H-TCM 199. After removal of cumulus cells the denuded oocytes were washed in fertilization medium three times and then fertilized with three different diluted semen (T22, T15 and M15) stored for different times (5 min, 24 h, 48 h or 72 h) before and after the incubation period (2 h at controlled temperature and atmosphere). For each semen sample, fertilization was performed in 50 ml drops with 1 106 sperm/ml at 39 8C with 5% CO2 in humidified air for 20 h. 2.4. In vitro culture (IVC) After IVF, the presumptive zygotes were washed in SOF to remove spermatozoa and cellular debris. The fertility rate was evaluated at this time by counting number of cleaved oocytes / number of matured oocytes. They were then divided into 20 ml culture drops (five to six embryos/drop) consisting of SOF supplemented with 1% (v/v) BME-essential amino acids (Gibco-Life Technologies BRL, Paisley, Scotland), 1% (v/v) MEM-nonessential amino acids (Gibco-Life Technologies BRL, Paisley, Scotland), 1 mM glutamine and 8 mg/ml fatty acid free bovine serum albumin (BSA). The incubation conditions were 7% O2, 5% CO2, and 88% N2 at 39 8C in humidified air. The culture was continued until 6–8 days after fertilization to evaluate the blastocyst rate (number of blastocysts/number of matured oocytes). 2.5. Statistical analysis This study was designed to compare different diluted semen (T15 versus T22, T15 versus M15, T22 versus M15) at different storage times (5 min, 24, 48, 72 h), by comparing their motility rate, motility score, fertilization rate and blastocyst rate parameters. Motility rate and motility score variables were compared using the GLM (general linear model) procedure by means of the SAS System (SAS/STAT User’s Guide, 6.03 edition, SAS). Fertilization rate and blastocyst rate variables were compared using the Chi-square test. A total of 150 semen samples were considered. Statistical significance was denoted as P < 0.05.
3. Results 3.1. Motility rate and motility score during storage The results obtained in this experiment are summarized in Table 1. Five minutes after storage all diluted semen samples had the same motility rate and motility score. After 24 h both the motility rate and the motility score were significantly
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Table 1 Motility rate and motility score of ram semen stored with three diluents and four different periods of storage Semen parameters
Diluents
Periods of storage 5 min
24 h
48 h
72 h
Motility rate (%)
T22 T15 M15
77.4 3.1a 77.4 3.1a 77.4 3.1a
55.3 3.1a 66.3 3.4b 41.4 2.8c
25.4 3.1d 35.5 3.4d 1.9 2.8e
9.0 3.4d 15.0 3.9d 0.1 2.9e
Motility score (0–5)
T22 T15 M15
3.5 0.1a 3.5 0.1a 3.5 0.1a
3.3 0.1a 3.5 0.1a 2.9 0.1b
2.3 0.1d 2.7 0.1d 0.5 0.1e
1.4 0.1d 1.9 0.2d 0.0e
Values in the same column with different superscripts differ:
a,b
P < 0.05;
a,c;b,c
P < 0.01;
d,e
P < 0.0001.
higher in the TEMPOL samples than in skimmed milk samples. The motility rate of T15 samples was significantly higher than T22 (P < 0.05), while the motility scores did not differ. After 48 and 72 h of storage, there were no significant differences in the motility rate and the motility scores among the TEMPOL samples, while these scores were very low in the skimmed milk samples, with highly significant differences (P < 0.0001) as compared with the TEMPOL samples. 3.2. In vitro fertilization Table 2 shows fertility and blastocyst rates. The fertilization rate was different for samples stored for different periods: after 5 min it was 80.0% for both T22 and T15, while for M15 it was 67.0% (no significant differences); after 24 h it was 64.3%, 76.5% and 64.5% for T22, T15 and M15 respectively, (no significant differences); after 48 h the fertility rate of T15 was 67.0%, significantly higher than those of the other samples (T2238.5%; M1518.9%) (P < 0.001); after 72 h fertility rates of 52.2%, 12.4% and 0% for T15, T22 and M15, respectively, were obtained, with significant differences between T15 and the other samples (P < 0.001). The blastocyst rate was likewise different for samples stored for different periods: after 5 min it was 40.0% for both T22 and T15, while for M15 it was 45.4% (no significant differences); after 24 h it was 39.7%, 43.5% and 47.9% for T22, T15 and M15, respectively Table 2 In vitro fertilization and blastocysts rate with semen stored in three diluents and four periods of storage Diluents
Periods of storage 5 min
24 h a
48 h a
72 h
Fertilization rate (%)
T22 T15 M15
80.0 (60/75) 80.0 (60/75)a 67.0 (59/88)a
64.3 (81/126) 76.5 (65/85)a 64.5 (78/121)a
38.5 (30/78) 67 (67/100)b 18.9 (14/74)c
12.4 (12/97)d 52.2 (24/46)e 0 (0/45)f
Blastocyst rate (%)
T22 T15 M15
40.0 (30/75)a 40.0 (30/75)a 45.4 (40/88)a
39.7 (50/126)a 43.5 (37/85)a 47.9 (58/121)a
20.5 (16/78)a 28.0 (28/100)a 4.0 (3/74)b
8.3 (6/72)g 21.7 (10/46)h 0 (0/45)i
Values in the same column with different superscripts differ: g,h;g,i P < 0.05.
a
a,b;b,c;a,c;d,e;e,f;h,i
P < 0.001,
d,f
P < 0.01,
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(no significant differences); after 48 h the blastocyst rate of T15 was 28.0%, and 20.5% for T22. The two TEMPOL samples did not differ significantly, but both the T15 and T22 samples had significantly higher blastocyst rates (P < 0.001) than the M15 sample (4%). After 72 h blastocyst rates were 21.7%, 8.3% and 0% for T15, T22 and M15, respectively, with significant differences between T15 and the other samples (P < 0.05). 3.3. Motility rate after 2 h of incubation at controlled temperature and low oxygen concentration After 2 h of incubation at controlled temperature and atmosphere, the semen samples (T22, T15 and M15) for each storage periods (5 min, 24, 48 and 72 h) had the following results (Fig. 1): samples stored for 5 min had a motility rate of 70.3% in T15, 69.7% in T22, and 59.6% in M15, with a statistically significant difference (P < 0.0001) between TEMPOL and skimmed milk samples. Samples stored for 24 h had motility rates of 49.6%, and 54.9% for T22 and T15 respectively, while in M15 only 2.8% of cells were motile, with a statistically significant difference between TEMPOL and skimmed milk samples (P < 0.0001). Samples stored for 48 h had motility rate of 9.8% for T22, 20.5% for T15 and 1.9% for M15, with a statistically significant difference (P < 0.0001) between TEMPOL and skimmed milk samples (P < 0.0001). Samples stored for 72 h had a very low motility rate: 5.5% and 10.4% for T22 and T15, respectively and 0% for M15, with a statistically significant difference (P < 0.0001) between TEMPOL and skimmed milk samples.
Fig. 1. Reduction of motility rate of ram semen stored with three diluents and four storage times after 2 h of incubation at controlled temperature and low oxygen concentration. Values in the different column with different superscripts differ: a,bP < 0.0001.
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Table 3 In vitro fertilization and blastocysts rate with semen stored in three diluents and four periods of storage after 2 h of incubation at controlled temperature and low oxygen concentration Diluents
Period of storage + 2 h incubation 5 min + 2 h
24 h + 2 h a
48 h + 2 h
72 h + 2 h
Fertilization rate (%)
T22 T15 M15
100 (36/36) 97.3 (36/37)a 37.5 (9/24)b
88.2 (45/51) 89.8 (44/49)a 0 (0/46)b
65.8 (27/41) 74.3 (29/39)a 0 (0/38)b
47.6 (20/42)a 71.8 (28/39)c 0 (0/37)b
Blastocyst rate (%)
T22 T15 M15
50.0 (18/36)a 54.0 (20/37)ab 20.8 (5/24)c
52.9 (27/51)a 53.1 (26/49)a 0 (0/46)b
34.1 (14/41)a 46.1 (18/39)a 0 (0/38)b
23.8 (10/42)a 38.5 (15/39)a 0 (0/37)b
Values in the same column with different superscripts differ:
a,b;b,c
a
a
P < 0.001;
a,c
P < 0.05.
3.4. In vitro fertilization after 2 h of semen incubation at controlled temperature and low oxygen concentration Table 3 shows the fertility and blastocyst rates after different incubation periods at controlled temperature and atmosphere. The fertilization rates were different for samples stored for different periods: after 5 min it was 100% for T22, 97.3% for T15 and 37.5% for M15, with significant differences between TEMPOL samples and M15 samples (P < 0.001); after 24 h it was 88.2%, 89.8% and 0% for T22, T15 and M15, respectively, with significant differences between TEMPOL and skimmed milk samples (P < 0.001); after 48 h it was 74.3% and 65.8% for T15 and T22 respectively, significantly higher than of the milk samples (M150%) (P < 0.001); after 72 h it was 71.8%, 47.6% and 0% for T15, T22 and M15, respectively, with significant differences between T15 and the other samples (P < 0.05 for T15 versus T22 and P < 0.001 for T15 versus M15). The blastocyst rate was different for samples stored for different periods: after 5 min it was 54.0%, 50.0% and 20.8% for T15, T22 and M15 respectively, with significant differences between T15 and M15 (P < 0.001) and between T22 and M15 (P < 0.05); after 24 h it was 52.9%, 53.1% and 0% for T22, T15 and M15, respectively, again with significant differences between the TEMPOL samples and M15 (P < 0.001); after 48 h it was 46.1% for T15 and 34.1% for T22. Both the T15 and the T22 samples had significantly higher blastocyst rates (P < 0.001) than the M15 sample (0%); after 72 h it was 38.5%, 23.8% and 0% for T15, T22 and M15, respectively, with significant differences between TEMPOL samples and skimmed milk samples (P < 0.001).
4. Discussion This study found that adding TEMPOL appears to protect stored ram semen by improving its motility and survival. Antioxidants are cytoplasmic enzyme systems found in every kind of cell. However sperm cells lose most of their cytoplasm during both spermatogenesis and preservation, and thus have a low content of these substances [35]. As a result, they become more susceptible to peroxidative processes which affect their survival and fertility. For this
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reason, to minimize damages, several authors added antioxidants during rabbit [19,36], bull [17,22,25,37], ram [6,26,38], turkey [29] and human semen storage [14–16,18]. TEMPOL is one of several antioxidants with beneficial effects on the motility and survival on bull semen [28]. Our results show that samples preserved with 2 mM of this antioxidant for 24, 48 and 72 h had higher motility rate and motility score than the control. Lindemann and Kanous [28] and Foote et al. [8] obtained similar results at the same concentration on chilled stored bull semen. Motility improvement was also demonstrated by Maxwell and Stojanov [26] and Upreti (38 in ram semen using different antioxidants). In our work, when the temperature was reduced to 15 8C, TEMPOL samples had a higher motility rate than at 22 8C. However, there were statistically significant differences only in samples stored for 24 h. In the samples stored for 24 h and 48 h, the decline of motility was very great, although even in this situation the T15 samples had the highest values. These results were similar to those previously described by Maxwell and Stojanov [26] on ram semen stored at different temperatures. The motility rate of these samples was similar to those of frozen-thawed semen used for intrauterine insemination. By contrast, motility was almost total absent in skimmed milk samples stored at 15 8C from 48 to 72 h. This shows that semen quality decreases in long-term storage. In fact, it is well known that ram semen diluted with skimmed milk and stored at 15 8C must be used between 8 and 10 h after storage [3]. Shannon and Curson [37] observed a higher catalase activity due to an increase in ROS caused by higher temperatures. Similarly, our results obtained by using TEMPOL at 15 8C and 22 8C might be affected by a higher semen storage temperature. It has been shown that 15 8C is the optimal temperature for maintain a good percentage of motile cells and a high motility score during short term storage [39]. A storage temperature for bull semen ranging between 15 8C and 27 8C gave a satisfactory calving rate [40]. Good motility does not necessarily correspond to a high fertility [41]. Indeed the membranes of preserved spermatozoa undergo changes similar to those observed during capacitation and acrosomal reactions and these could make spermatozoa infertile [4]. Nevertheless, in this preliminary study, motility did correspond to good fertility for all samples within the first 24 h of storage. TEMPOL 15 samples had the highest fertility and blastocyst rates, despite the overall low motility among all the samples stored for more than 24 h, probably due to its protective effect against membrane peroxidation. This supports Alvarez and Storey’s results that superoxide dismutase plays an important role in protecting mammalian sperm from oxygen toxicity [19]. Thompson et al. [42] reported that in sheep oviduct the pO2 tension is approximately 60 mmHg, corresponding to an oxygen concentration in the lumen of between 6% and 10%. For this reason, semen samples preserved for up to three days were incubated at controlled temperature and low oxygen concentration to test for the effects of low pO2 on their survival. The results of the incubation trials showed that TEMPOL samples maintained a good motility rate for at least 24 h after storage. By contrast, there was already a total decline in the motility rate of skimmed milk samples 24 h after storage. Mammalian spermatozoa, matured in the epididymis and ejaculated, are not able to fertilize oocytes and they undergo some physiological changes in the female genital tract, thus becoming capacitated. For this reason, there is always some delay between
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insemination and the beginning of fertilization. This delay can be less than 1h or more than several hours, depending on the species [43]. Furthermore, in the case of the sheep, capacitation might be much more efficient at temperature slightly higher than 38 8C [43]. Previous papers suggest that a period of 2 h of incubation it is necessary to induce in vitro capacitation of ram semen [34,44,45]. Thus, the higher fertility and blastocyst rates 2 h after incubation observed in this paper might be due to the semen acquiring the capacity to fertilize oocytes. In conclusion, semen preserved with TEMPOL at 15 8C for the four different periods of storage maintained better motility, fertility and blastocyst rate before and after 2 h of incubation than did TEMPOL at 22 8C. Thus, TEMPOL is a stronger antioxidant at temperatures below room temperature and it seems that its effectiveness is due to a combination of temperature and antioxidant effect. Furthermore when TEMPOL is compared to skim milk the benefits are readily apparent. These results lead us to hypothesise that lowering the temperatures still more could have a positive effect on ram semen preserved for longer periods. However, in vivo trials are necessary in order to evaluate the fertility and lambing rate after cervical insemination of ram semen stored with TEMPOL.
Acknowledgments The authors thank Mr. G. Camoglio and Mr. A. Pintadu for expert management of the animals.
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[12] VanDemark NL, Salisbury GW. Oxygen damage to bull spermatozoa and its prevention by catalase. J Dairy Sci 1949;32:353–60. [13] Foote RH. Influence of light and agitation on bovine spermatozoa stored with protective agents. J Dairy Sci 1967;50:1468–74. [14] Aitken RJ, Clarkson JS. Significance of reactive species and antioxidants in defining the efficacy of sperm preparative techniques. J Androl 1989;9:367–76. [15] Storey BT. Biochemistry of the induction and prevention of lipoperoxidative damage in human spermatozoa. Mol Hum Reprod 1997;3:203–14. [16] Aitken RJ, Gordon E, Harkiss D, Twigg JP, Milne P, Jennings Z, et al. Relative impact of oxidative stress on the functional competence and genomic integrity of human spermatozoa. Biol Reprod 1998;59:1037–46. [17] Bilodeau JF, Blanchette S, Gagnon C, Sirard MA. Thiols prevent H2O2-mediated loss of sperm motility in cryopreserved bull semen. Theriogenology 2001;56:275–86. [18] Alvarez JG, Touchstone JC, Blasco L, Stirey B. Spontaneus lipid peroxidation and production of hydrogen peroxide and superoxide in human spermatozoa. J Androl 1987;8:338–48. [19] Alvarez JG, Storey BT. Role of superoxide dismutase in protecting rabbit spermatozoa from O2 toxicity due to lipid peroxidation. Biol Reprod 1983;28:1129–36. [20] Alvarez JG, Storey BT. Taurine, hypotaurine, epinephrine and albumin inhibit lipid peroxidation in rabbit spermatozoa and protect against loss of motility. Biol Reprod 1983;29:548–55. [21] Beconi MT, Francia CR, Mora NG, Affranchino MA. Effect of natural antioxidants on frozen bovine semen preservation. Theriogenology 1993;40:841–51. [22] Foote RH. Motility and fertility of bull semen extender at high rate in yolk extender containing catalase. J Dairy Sci 1962;45:1237–41. [23] Foote RH. Survival of bull sperm in milk and yolk extenders with added catalase. J Dairy Sci 1962;76:1233– 47. [24] LaPointe S, Sirard MA. Catalase and oviduct fluid reverse the decreased motility of bovine sperm in culture medium containing specific amino acids. J Androl 1998;19:31–6. [25] Lindemann CB, O’Brein JA, Giblin FJ. An investigation of the effectiveness of certain antioxidants in preserving the motility of reactivated bull sperm models. Biol Reprod 1988;38:114–20. [26] Maxwell WMC, Stojanov T. Liquid storage of ram semen in the absence or presence of some antioxidants. Reprod Fertil Dev 1996;8:1013–20. [27] Mitchell J. Biologically active metal-independent superoxide dismutase mimics. Biochemistry 1990;29:2802–7. [28] Lindemann CB, Kanous K. The cyclic nitroxide free radical, 4-hidroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), is effective in prolonging the motility of bull sperm in vitro. Biol Reprod 1991;44(Suppl 1):117 [abstract]. [29] Donoghue AM, Donoghue DJ. Effects of water- and lipid-soluble antioxidants on turkey sperm viability, membrane integrity, and motility during liquid storage. Poult Sci 1997;76:1440–5. [30] Upreti GC, Oliver JE, Duganzich DM, Munday R, Smith JF. Development of a chemically defined ram semen diluent (RSD-1). Anim Reprod Sci 1995;37:143–57. [31] Evans G, Maxwell WMC. Semen and its characteristics. In: Salamon’s artificial insemination of sheep and goats. Sidney: Butterworths; 1987. 22–30. [32] De Matos DG, Furnus CC, Moses DF, Baldassarre H. Effect of cysteamine on glutathione level and developmental capacity of bovine oocytes matured in vitro. Mol Reprod Dev 1995;42:432–6. [33] Ptak G, Dattena M, Loi P, Tischner M, Cappai P. Ovum pick-up: efficiency of in vitro embryo production, vitrification and birth of offspring. Theriogenology 1999;52:1105–14. [34] Accardo C, Dattena M, Pilichi S, Mara L, Chessa B, Cappai P. Effect of recombinant human FSH and LH on in vitro maturation of sheep oocytes; embryo development and viability. Anim Reprod Sci 2004;81:77–86. [35] Li T-K. The glutathione and thiol content of mammalian spermatozoa and seminal plasma. Biol Reprod 1975;12:641–6. [36] Alvarez JG, Storey BT. Role of glutathione peroxidase in protecting mammalian spermatozoa from loss of motility caused by spontaneous lipid peroxidation. Gamete Res 1989;23:77–90.
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[37] Shannon P, Curson B. Kinetics of the aromatic L-amino acid oxidase from dead bovine spermatozoa and effect of catalase on fertility of diluted bovine semen stored at 5 8C and ambient temperatures. J Reprod Fertil 1982;64:463–7. [38] Upreti GC, Jensen K, Oliver JE, Duganzich DM, Munday R, Smith JF. Motility of ram spermatozoa during storage in a chemically defined diluent containing antioxidants. Anim Reprod Sci 1997;48:269–78. [39] Upreti GC. Effect of physical parameters on ram spermatozoal motility. Proc N Zeal Soc Anim Prod 1992;52. [40] Shannon P. Factors affecting semen preservation and concentration rates in cattle. J Reprod Fertil 1978;54:519–27. [41] Windsor DP. Mitochondrial function and ram sperm fertility. Reprod Fertil Dev 1997;9:279–84. [42] Thompson JGE, Simpson AC, Pugh PA, Donnely PE, Tervit HR. Effect of oxygen on in vitro development of preimplantation sheep and goats embryos. J Reprod Fertil 1990;89:573–8. [43] Yanagimachi R. In: Knobil E, Neill J, editors. et al. The physiology of reproduction. New York: Raven Press, Ltd.; 1988. p. 135–85. [44] Pe´ rez LJ, Valca´ rcel A, de las Heras MA, Moses DF, Baldassarre H. In vitro capacitation and induction of acrosomal exocytosis in ram spermatozoa as assessed by the chlortetracycline assay. Theriogenology 1997;45:1037–46. [45] de las Heras MA, Valcarcel A, Perez LJ. In vitro capacitation effect of gamma-amminobutyric acid in ram spermatozoa. Biol Reprod 1997;56:964–8.
Benefits of TEMPOL on ram semen motility and in vitro fertility: a preliminary study Laura Maraa,*, Carla Accardoa, Susanna Pilichia, Maria Dattenaa, Fabrizio Chessaa, Bernardo Chessab, Andrea Brancaa, Pietro Cappaia a Istituto Zootecnico e Caseario per la Sardegna, 07040 Olmedo (Sassari), Italy Istituto di Patologia Speciale e Clinica Medica Veterinaria, Facolta` di Medicina Veterinaria, via Vienna, 07100 Sassari, Italy
b
Received 7 July 2004; received in revised form 28 September 2004; accepted 7 October 2004
Abstract Extending the preservation time of fresh semen is an important goal in artificial insemination programs particularly for ewes in natural oestrus, where insemination periods are longer than for ewes synchronized with hormonal treatments. The aim of this study was to evaluate the effect of the antioxidant TEMPOL (4-hydroxy-2,2,6,6tetramethylpiperidine-1-oxyl) on the maintenance in long term storage of ram semen motility and fertility. Semen from Sarda breed rams was diluted in two extenders: sodium citrate buffer with TEMPOL and skimmed milk, used as control. Samples diluted with TEMPOL were cooled at either 15 8C or 22 8C, while those diluted with skimmed milk were cooled at 15 8C. Each sample was divided into four stocks, and stored for different times (5 min, 24, 48 and 72 h). Three aliquots were taken from each stock for every storage period. One was immediately evaluated under microscope; one was used for in vitro fertilization; one was incubated for 2 h in controlled humidified atmosphere (5% CO2, 7% O2 and 88% N2) at 39 8C, then evaluated for motility and utilized for in vitro fertilization. Ram semen diluted with media containing TEMPOL demonstrated increased motility, fertility and an improved protective effect when it was stored at 15 8C. # 2004 Elsevier Inc. All rights reserved. Keywords: Semen storage; Antioxidant; Motility; Fertility; In vitro fertilization * Corresponding author. Tel.: +39 079 387 268; fax: +39 079 389 450. E-mail address: [email protected] (L. Mara). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.10.005
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1. Introduction Sheep reproduction is highly seasonal. Hormones are currently used to extend the sexual activity of females outside the natural lambing season, although consumers are beginning to react negatively to their use. Several projects are trying to develop alternatives to hormone therapy in the field of small ruminant reproduction. However, if hormones are not used to synchronize females, ovulation will take place over a longer period, and the delay between sperm collection and fertilization will be more variable. Using stored frozen or liquid semen is one of several strategies that may be used to overcome this problem. However, the use of frozen-thawed semen is limited by its relatively short-term fertility and requires employing the laborious intrauterine insemination technique, which is not easy to perform under field conditions. The practical and economic advantages of using liquid chilled semen are clear. It is cheap, easy to handle and can be used in cervical insemination. For the last ten years fresh ram semen diluted with skimmed milk and stored at 15 8C has been the most widely used method in AI programs in the Sarda breed. The fertility rate is about 50% [1]. However this semen needs to be used within 8–10 h after dilution to guarantee good fertility [2,3]. In recent years alternative methods have been suggested to improve preservation of semen quality during storage [4–7]. Recently, Foote et al. [8] found that adding antioxidants is beneficial for sperm stored for long periods. Sperm, unlike other types of cells, have a low content of substances [8,9] to protect them against the destructive effect of reactive oxygen species (ROS) [10]. Moreover, the unsaturated fatty acids content in their membranes is high. This makes them more susceptible to peroxidative damage [11]. During manipulation before storage, sperm is exposed to oxygen and visible light radiation and this may lead to ROS formation and resulting damage to sperm cell motility and genomic integrity [12–17]. In these conditions antioxidants could help to maintain sperm motility and survival [18–26]. Recent studies show that TEMPOL, a stable membrane-permeable nitroxide with SODlike activity [27], can increase in vitro sperm survival in bulls [8,28] and turkeys [29]. The aim of this study was to investigate the benefits for ram semen motility of this antioxidant under long-term storage conditions and at different temperatures. The effect of TEMPOL on semen used for in vitro fertilization of sheep oocytes is also reported.
2. Materials and methods Except where otherwise indicated, all chemicals were obtained from the Sigma (St. Louis, MO, USA). 2.1. Collection, dilution and assessment of semen Ten Sarda breed adult rams (3–6 years old), of proven high fertility were used in the experiment. Each ram was used on alternate days. Semen was collected once a day between 8:00 and 10:00 a.m. by artificial vagina. Two ejaculates were collected per session and immediately placed in a water-bath at 30 8C. The percentage of motile sperm was
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estimated under phase-contrast microscopy at 38 8C on a hot plate, while the optical density of the sperm concentration was evaluated using a calibrated spectrophotometer. Only ejaculates with an acceptable motility score higher than 3 (on a scale of 0–5, where a score of 5 indicates rapid swirling motion and 0 denotes absence of motion) and with a spermatozoa concentration higher than 3 109 ml 1 were used. Ejaculates were diluted at a concentration of 1.6 109 spermatozoa/ml at 30 8C, employing two different extenders: sodium citrate buffer (0.1 M + 5 mM MgSO4 + 2 mM fructose) [27] added with 2 mM TEMPOL (4-hydroxy 2,2,6,6-tetramethylpiperidine) [25] or skimmed milk (90 ml sterile H2O mQ + 300 mg sulphanilamide + 10 g skim milk powderOxoid (UK) + 100 mg streptomycinsulfat + 100.000 UI penicillin G) [2] used as a control. Skimmed milk is the medium routinely used in our laboratory to dilute fresh ram semen. Five minutes after dilution, semen motility was evaluated under a phase contrast microscope by placing a 10 ml drop of diluted semen on a slide covered with a glass coverslip (22 mm 22 mm). Motility evaluation was performed by focusing the binocular phase-contrast microscope at the centre of the coverslip at 250 magnification. Slide temperature was maintained at 38 8C by using a heating stage fitted to the microscope [30]. The motility rate was determined by estimating the proportion of motile and non-motile cells, while the motility score was based on the individual spermatozoa movements, using an arbitrary scale ranging from 0 to 5, as described by Evans and Maxwell [31]. Samples diluted with TEMPOL were cooled to either 15 8C (T15) or 22 8C (T22), while all those diluted with skimmed milk were cooled at 15 8C (M15). Cooling was performed as follows: to reach 15 8C, diluted semen was placed in a beaker containing water at 30 8C and a glass ampoule of glacial acetic acid. This in turn was placed in a cold storage at 15 8C. Samples cooled to 22 8C were simply left at room temperature (22 8C). Each sample was then divided in four stocks, stored for 5 min, 24, 48 and 72 h, respectively. Three aliquots of 250 ml each were taken from each stock: one was resuspended in 2 ml of its storage liquid and evaluated under microscope; another aliquot was used directly for IVF; the last one was incubated for 2 h at the following incubation conditions: 7% O2, 5% CO2 and 88% N2 at 39 8C in humidified air. Sperm motility was evaluated and the sample was then used for IVF. 2.2. Oocytes collection and maturation Sheep ovaries were obtained from a slaughterhouse and within 2–3 h were transported to the laboratory in sterile physiological saline solution at approximately 35 8C. Oocytes were collected by aspiration of the follicles (2–6 mm in diameter) with a 5 ml syringe fitted with a 20-gauge needle and placed in tissue culture medium (TCM-199) supplemented with 5% calf serum, 0.05 mg/ml heparin and 0.05 mg/ml gentamicin sulphate. Oocytes were washed in Hepes-buffered TCM 199 (H-TCM 199) supplemented with 0.4% BSA (bovine serum albumin fraction V, A 9418), and 2 mM glutamine. The medium used for maturation was bicarbonate-buffered TCM 199 with the osmolarity adjusted to 275 mOsm and containing 2 mM glutamine, 5 mg/ml follicle stimulating hormone (FSH) (Ovagen ICP, Auckland, NZ), 5 mg/ml luteinizing hormone (LH), 1 mg/ml estradiol, 0.3 mM sodium pyruvate and 100 mM cysteamine [32]. The oocytes were incubated in
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400 ml of medium in 4-well dishes (Nunc, Nunclon, Denmark) with each well containing 20–30 oocytes covered with mineral oil. All dishes were incubated at 39 8C in air in a humidified atmosphere of 5% CO2 for 24 h [33,34]. 2.3. In vitro fertilization (IVF) Following maturation, oocytes were partially stripped of surrounding cumulus cells using a fine pipette and 300 UI/ml of hyaluronidase in H-TCM 199. After removal of cumulus cells the denuded oocytes were washed in fertilization medium three times and then fertilized with three different diluted semen (T22, T15 and M15) stored for different times (5 min, 24 h, 48 h or 72 h) before and after the incubation period (2 h at controlled temperature and atmosphere). For each semen sample, fertilization was performed in 50 ml drops with 1 106 sperm/ml at 39 8C with 5% CO2 in humidified air for 20 h. 2.4. In vitro culture (IVC) After IVF, the presumptive zygotes were washed in SOF to remove spermatozoa and cellular debris. The fertility rate was evaluated at this time by counting number of cleaved oocytes / number of matured oocytes. They were then divided into 20 ml culture drops (five to six embryos/drop) consisting of SOF supplemented with 1% (v/v) BME-essential amino acids (Gibco-Life Technologies BRL, Paisley, Scotland), 1% (v/v) MEM-nonessential amino acids (Gibco-Life Technologies BRL, Paisley, Scotland), 1 mM glutamine and 8 mg/ml fatty acid free bovine serum albumin (BSA). The incubation conditions were 7% O2, 5% CO2, and 88% N2 at 39 8C in humidified air. The culture was continued until 6–8 days after fertilization to evaluate the blastocyst rate (number of blastocysts/number of matured oocytes). 2.5. Statistical analysis This study was designed to compare different diluted semen (T15 versus T22, T15 versus M15, T22 versus M15) at different storage times (5 min, 24, 48, 72 h), by comparing their motility rate, motility score, fertilization rate and blastocyst rate parameters. Motility rate and motility score variables were compared using the GLM (general linear model) procedure by means of the SAS System (SAS/STAT User’s Guide, 6.03 edition, SAS). Fertilization rate and blastocyst rate variables were compared using the Chi-square test. A total of 150 semen samples were considered. Statistical significance was denoted as P < 0.05.
3. Results 3.1. Motility rate and motility score during storage The results obtained in this experiment are summarized in Table 1. Five minutes after storage all diluted semen samples had the same motility rate and motility score. After 24 h both the motility rate and the motility score were significantly
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Table 1 Motility rate and motility score of ram semen stored with three diluents and four different periods of storage Semen parameters
Diluents
Periods of storage 5 min
24 h
48 h
72 h
Motility rate (%)
T22 T15 M15
77.4 3.1a 77.4 3.1a 77.4 3.1a
55.3 3.1a 66.3 3.4b 41.4 2.8c
25.4 3.1d 35.5 3.4d 1.9 2.8e
9.0 3.4d 15.0 3.9d 0.1 2.9e
Motility score (0–5)
T22 T15 M15
3.5 0.1a 3.5 0.1a 3.5 0.1a
3.3 0.1a 3.5 0.1a 2.9 0.1b
2.3 0.1d 2.7 0.1d 0.5 0.1e
1.4 0.1d 1.9 0.2d 0.0e
Values in the same column with different superscripts differ:
a,b
P < 0.05;
a,c;b,c
P < 0.01;
d,e
P < 0.0001.
higher in the TEMPOL samples than in skimmed milk samples. The motility rate of T15 samples was significantly higher than T22 (P < 0.05), while the motility scores did not differ. After 48 and 72 h of storage, there were no significant differences in the motility rate and the motility scores among the TEMPOL samples, while these scores were very low in the skimmed milk samples, with highly significant differences (P < 0.0001) as compared with the TEMPOL samples. 3.2. In vitro fertilization Table 2 shows fertility and blastocyst rates. The fertilization rate was different for samples stored for different periods: after 5 min it was 80.0% for both T22 and T15, while for M15 it was 67.0% (no significant differences); after 24 h it was 64.3%, 76.5% and 64.5% for T22, T15 and M15 respectively, (no significant differences); after 48 h the fertility rate of T15 was 67.0%, significantly higher than those of the other samples (T2238.5%; M1518.9%) (P < 0.001); after 72 h fertility rates of 52.2%, 12.4% and 0% for T15, T22 and M15, respectively, were obtained, with significant differences between T15 and the other samples (P < 0.001). The blastocyst rate was likewise different for samples stored for different periods: after 5 min it was 40.0% for both T22 and T15, while for M15 it was 45.4% (no significant differences); after 24 h it was 39.7%, 43.5% and 47.9% for T22, T15 and M15, respectively Table 2 In vitro fertilization and blastocysts rate with semen stored in three diluents and four periods of storage Diluents
Periods of storage 5 min
24 h a
48 h a
72 h
Fertilization rate (%)
T22 T15 M15
80.0 (60/75) 80.0 (60/75)a 67.0 (59/88)a
64.3 (81/126) 76.5 (65/85)a 64.5 (78/121)a
38.5 (30/78) 67 (67/100)b 18.9 (14/74)c
12.4 (12/97)d 52.2 (24/46)e 0 (0/45)f
Blastocyst rate (%)
T22 T15 M15
40.0 (30/75)a 40.0 (30/75)a 45.4 (40/88)a
39.7 (50/126)a 43.5 (37/85)a 47.9 (58/121)a
20.5 (16/78)a 28.0 (28/100)a 4.0 (3/74)b
8.3 (6/72)g 21.7 (10/46)h 0 (0/45)i
Values in the same column with different superscripts differ: g,h;g,i P < 0.05.
a
a,b;b,c;a,c;d,e;e,f;h,i
P < 0.001,
d,f
P < 0.01,
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(no significant differences); after 48 h the blastocyst rate of T15 was 28.0%, and 20.5% for T22. The two TEMPOL samples did not differ significantly, but both the T15 and T22 samples had significantly higher blastocyst rates (P < 0.001) than the M15 sample (4%). After 72 h blastocyst rates were 21.7%, 8.3% and 0% for T15, T22 and M15, respectively, with significant differences between T15 and the other samples (P < 0.05). 3.3. Motility rate after 2 h of incubation at controlled temperature and low oxygen concentration After 2 h of incubation at controlled temperature and atmosphere, the semen samples (T22, T15 and M15) for each storage periods (5 min, 24, 48 and 72 h) had the following results (Fig. 1): samples stored for 5 min had a motility rate of 70.3% in T15, 69.7% in T22, and 59.6% in M15, with a statistically significant difference (P < 0.0001) between TEMPOL and skimmed milk samples. Samples stored for 24 h had motility rates of 49.6%, and 54.9% for T22 and T15 respectively, while in M15 only 2.8% of cells were motile, with a statistically significant difference between TEMPOL and skimmed milk samples (P < 0.0001). Samples stored for 48 h had motility rate of 9.8% for T22, 20.5% for T15 and 1.9% for M15, with a statistically significant difference (P < 0.0001) between TEMPOL and skimmed milk samples (P < 0.0001). Samples stored for 72 h had a very low motility rate: 5.5% and 10.4% for T22 and T15, respectively and 0% for M15, with a statistically significant difference (P < 0.0001) between TEMPOL and skimmed milk samples.
Fig. 1. Reduction of motility rate of ram semen stored with three diluents and four storage times after 2 h of incubation at controlled temperature and low oxygen concentration. Values in the different column with different superscripts differ: a,bP < 0.0001.
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Table 3 In vitro fertilization and blastocysts rate with semen stored in three diluents and four periods of storage after 2 h of incubation at controlled temperature and low oxygen concentration Diluents
Period of storage + 2 h incubation 5 min + 2 h
24 h + 2 h a
48 h + 2 h
72 h + 2 h
Fertilization rate (%)
T22 T15 M15
100 (36/36) 97.3 (36/37)a 37.5 (9/24)b
88.2 (45/51) 89.8 (44/49)a 0 (0/46)b
65.8 (27/41) 74.3 (29/39)a 0 (0/38)b
47.6 (20/42)a 71.8 (28/39)c 0 (0/37)b
Blastocyst rate (%)
T22 T15 M15
50.0 (18/36)a 54.0 (20/37)ab 20.8 (5/24)c
52.9 (27/51)a 53.1 (26/49)a 0 (0/46)b
34.1 (14/41)a 46.1 (18/39)a 0 (0/38)b
23.8 (10/42)a 38.5 (15/39)a 0 (0/37)b
Values in the same column with different superscripts differ:
a,b;b,c
a
a
P < 0.001;
a,c
P < 0.05.
3.4. In vitro fertilization after 2 h of semen incubation at controlled temperature and low oxygen concentration Table 3 shows the fertility and blastocyst rates after different incubation periods at controlled temperature and atmosphere. The fertilization rates were different for samples stored for different periods: after 5 min it was 100% for T22, 97.3% for T15 and 37.5% for M15, with significant differences between TEMPOL samples and M15 samples (P < 0.001); after 24 h it was 88.2%, 89.8% and 0% for T22, T15 and M15, respectively, with significant differences between TEMPOL and skimmed milk samples (P < 0.001); after 48 h it was 74.3% and 65.8% for T15 and T22 respectively, significantly higher than of the milk samples (M150%) (P < 0.001); after 72 h it was 71.8%, 47.6% and 0% for T15, T22 and M15, respectively, with significant differences between T15 and the other samples (P < 0.05 for T15 versus T22 and P < 0.001 for T15 versus M15). The blastocyst rate was different for samples stored for different periods: after 5 min it was 54.0%, 50.0% and 20.8% for T15, T22 and M15 respectively, with significant differences between T15 and M15 (P < 0.001) and between T22 and M15 (P < 0.05); after 24 h it was 52.9%, 53.1% and 0% for T22, T15 and M15, respectively, again with significant differences between the TEMPOL samples and M15 (P < 0.001); after 48 h it was 46.1% for T15 and 34.1% for T22. Both the T15 and the T22 samples had significantly higher blastocyst rates (P < 0.001) than the M15 sample (0%); after 72 h it was 38.5%, 23.8% and 0% for T15, T22 and M15, respectively, with significant differences between TEMPOL samples and skimmed milk samples (P < 0.001).
4. Discussion This study found that adding TEMPOL appears to protect stored ram semen by improving its motility and survival. Antioxidants are cytoplasmic enzyme systems found in every kind of cell. However sperm cells lose most of their cytoplasm during both spermatogenesis and preservation, and thus have a low content of these substances [35]. As a result, they become more susceptible to peroxidative processes which affect their survival and fertility. For this
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reason, to minimize damages, several authors added antioxidants during rabbit [19,36], bull [17,22,25,37], ram [6,26,38], turkey [29] and human semen storage [14–16,18]. TEMPOL is one of several antioxidants with beneficial effects on the motility and survival on bull semen [28]. Our results show that samples preserved with 2 mM of this antioxidant for 24, 48 and 72 h had higher motility rate and motility score than the control. Lindemann and Kanous [28] and Foote et al. [8] obtained similar results at the same concentration on chilled stored bull semen. Motility improvement was also demonstrated by Maxwell and Stojanov [26] and Upreti (38 in ram semen using different antioxidants). In our work, when the temperature was reduced to 15 8C, TEMPOL samples had a higher motility rate than at 22 8C. However, there were statistically significant differences only in samples stored for 24 h. In the samples stored for 24 h and 48 h, the decline of motility was very great, although even in this situation the T15 samples had the highest values. These results were similar to those previously described by Maxwell and Stojanov [26] on ram semen stored at different temperatures. The motility rate of these samples was similar to those of frozen-thawed semen used for intrauterine insemination. By contrast, motility was almost total absent in skimmed milk samples stored at 15 8C from 48 to 72 h. This shows that semen quality decreases in long-term storage. In fact, it is well known that ram semen diluted with skimmed milk and stored at 15 8C must be used between 8 and 10 h after storage [3]. Shannon and Curson [37] observed a higher catalase activity due to an increase in ROS caused by higher temperatures. Similarly, our results obtained by using TEMPOL at 15 8C and 22 8C might be affected by a higher semen storage temperature. It has been shown that 15 8C is the optimal temperature for maintain a good percentage of motile cells and a high motility score during short term storage [39]. A storage temperature for bull semen ranging between 15 8C and 27 8C gave a satisfactory calving rate [40]. Good motility does not necessarily correspond to a high fertility [41]. Indeed the membranes of preserved spermatozoa undergo changes similar to those observed during capacitation and acrosomal reactions and these could make spermatozoa infertile [4]. Nevertheless, in this preliminary study, motility did correspond to good fertility for all samples within the first 24 h of storage. TEMPOL 15 samples had the highest fertility and blastocyst rates, despite the overall low motility among all the samples stored for more than 24 h, probably due to its protective effect against membrane peroxidation. This supports Alvarez and Storey’s results that superoxide dismutase plays an important role in protecting mammalian sperm from oxygen toxicity [19]. Thompson et al. [42] reported that in sheep oviduct the pO2 tension is approximately 60 mmHg, corresponding to an oxygen concentration in the lumen of between 6% and 10%. For this reason, semen samples preserved for up to three days were incubated at controlled temperature and low oxygen concentration to test for the effects of low pO2 on their survival. The results of the incubation trials showed that TEMPOL samples maintained a good motility rate for at least 24 h after storage. By contrast, there was already a total decline in the motility rate of skimmed milk samples 24 h after storage. Mammalian spermatozoa, matured in the epididymis and ejaculated, are not able to fertilize oocytes and they undergo some physiological changes in the female genital tract, thus becoming capacitated. For this reason, there is always some delay between
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insemination and the beginning of fertilization. This delay can be less than 1h or more than several hours, depending on the species [43]. Furthermore, in the case of the sheep, capacitation might be much more efficient at temperature slightly higher than 38 8C [43]. Previous papers suggest that a period of 2 h of incubation it is necessary to induce in vitro capacitation of ram semen [34,44,45]. Thus, the higher fertility and blastocyst rates 2 h after incubation observed in this paper might be due to the semen acquiring the capacity to fertilize oocytes. In conclusion, semen preserved with TEMPOL at 15 8C for the four different periods of storage maintained better motility, fertility and blastocyst rate before and after 2 h of incubation than did TEMPOL at 22 8C. Thus, TEMPOL is a stronger antioxidant at temperatures below room temperature and it seems that its effectiveness is due to a combination of temperature and antioxidant effect. Furthermore when TEMPOL is compared to skim milk the benefits are readily apparent. These results lead us to hypothesise that lowering the temperatures still more could have a positive effect on ram semen preserved for longer periods. However, in vivo trials are necessary in order to evaluate the fertility and lambing rate after cervical insemination of ram semen stored with TEMPOL.
Acknowledgments The authors thank Mr. G. Camoglio and Mr. A. Pintadu for expert management of the animals.
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