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Effect of storage duration, storage temperature, and diluent on the viability and fertility of fresh ram sperm
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Theriogenology 73 (2010) 541–549 www.theriojournal.com
Effect of storage duration, storage temperature, and diluent on the viability and fertility of fresh ram sperm L. O’Hara a,c, J.P. Hanrahan a, L. Richardson a,c, A. Donovan a, S. Fair b, A.C.O. Evans c, P. Lonergan c,* a Teagasc, Animal Production Research Centre, Athenry, Ireland Physiology, School of Medicine, National University of Ireland Galway, Galway, Ireland c School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Ireland b
Received 20 August 2009; received in revised form 3 October 2009; accepted 6 October 2009
Abstract Cervical artificial insemination (AI) in sheep with fresh semen yields a much higher pregnancy rate than when frozen-thawed semen is used, and consequently frozen semen is only acceptable for laparoscopic insemination. The short life span of fresh semen is a major constraint on the use of AI in genetic improvement programs for sheep. The main objective of this study was to examine the effects of storage conditions on viability and fertilization ability of fresh ram (Ovis aries) semen up to 72 h postcollection. Experiment 1 was designed to evaluate the effect of diluent type (standard skim milk, AndroMed, OviPro, and INRA 96) and storage temperature (5 8C and 15 8C) on the motility and viability of fresh ram semen. Storage temperature, irrespective of diluent, had a significant effect on both motility and viability. Storage at 5 8C maintained acceptable motility and viability up to 72 h compared with that of storage at 15 8C. In Experiment 2, the penetrating ability of fresh ram semen, diluted in either skim milk, AndroMed, or INRA 96, was assessed using artificial mucus. Flat capillary tubes containing artificial mucus were suspended in 250 mL semen at a sperm concentration of 20 106/ mL. Semen was stored at 5 8C and tested after 6, 24, 48, and 72 h. There was a significant diluent by time interaction. In Experiment 3, the fertilizing ability of fresh ram semen stored at 5 8C was evaluated in vitro. Fresh semen (diluted in either skim milk, AndroMed, or INRA 96) was added to matured ewe oocytes at 6, 24, or 72 h after semen collection. Cleavage rate was recorded at 48 h postinsemination, and blastocyst development was recorded on Days 6 to 9. There was a significant treatment effect on cleavage and blastocyst rates; insemination of semen stored for 24 h resulted in higher rates than those for storage at 72 h. In Experiment 4, the fertilizing ability of fresh ram semen was evaluated in vivo. Semen was diluted in INRA 96, stored at 5 8C, and used to inseminate ewes on the day of collection or at 24, 48, and 72 h postcollection. Multiparous ewes were cervically inseminated at a synchronized estrus. Fertility rate decreased linearly (P < 0.001) up to 72 h after semen collection. # 2010 Elsevier Inc. All rights reserved. Keywords: AI; IVF; Semen extender; Sheep
1. Introduction Artificial insemination (AI), when used in conjunction with accurate progeny testing schemes, can * Corresponding author. Tel.: +353 1 6012147; fax: +353 1 6288421. E-mail address: [email protected] (P. Lonergan). 0093-691X/$ – see front matter # 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.10.009
substantially increase the rate of genetic progress compared with that of natural service and is the method of choice for dissemination of genetic improvement. Artificial insemination is an essential and wellestablished facilitator of genetic improvement in cattle, but the absence of an equally effective technology in sheep, where it would lead to increased accuracy of across-flock evaluation and higher selection intensity,
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represents a significant constraint for genetic improvement programs [1]. In sheep, whereas cervical AI with fresh semen yields an acceptable pregnancy rate, the short shelf life (10 to 14 h) of fresh semen [2] coupled with the natural limitation on the number of semen doses that can be obtained per ram per unit time plus small flock sizes seriously restricts the widespread use of individual sires. Several extenders have been developed for fresh storage of semen in cattle but, to date, none is capable of storing sperm for more than 2 to 3 d without a drop in fertility [3]; the situation is much worse in sheep. The widespread use of AI and the realization of its full potential depend on the use of frozen-thawed semen [4]. However, AI with frozen-thawed ram semen has not been widely adopted for sheep mainly due to the very poor fertility obtained after cervical insemination compared with the pregnancy rate after cervical insemination with fresh semen or that obtained with laparoscopic intrauterine insemination [4–7]. The low fertility from cervical insemination with frozen-thawed semen is attributed to irreversible damage caused to the cells during the freeze-thaw process [8–12]. Of the high proportion (40% to 60%) of ram spermatozoa that maintain their motility after freeze thawing, only 20% to 30% may remain biologically undamaged and therefore capable of successful fertilization; the few remaining undamaged spermatozoa are then often unable to transverse the cervical barrier and female reproductive tract to the site of fertilization (reviewed by Salamon and Maxwell [13]). Evidence from our group would support this notion; thus, we have shown that pregnancy rate after laparoscopic AI using frozenthawed semen is higher than that after cervical AI [14] and in addition that the number of accessory sperm on the zona pellucida is significantly higher than that after cervical AI, suggesting a problem with sperm transport to the site of fertilization [14]. However, laparoscopic AI is expensive, requires technical expertise, and raises concerns for animal welfare and thus is not likely to be a viable long-term alternative to the development of strategies for improving pregnancy rate to cervical AI. Storage diluents and techniques have been developed and adapted with the aim of improving the cryopreservation of ram semen [15–18], but fertility after cervical AI with frozen-thawed sperm still remains significantly lower than that achieved with fresh semen. Therefore, cervical AI with fresh semen would be a more attractive option especially if the effective length of fresh semen could be extended to 48 h. This would represent a major improvement on current practice and would have several advantages including (i) bypassing
problems of poor fertility associated with frozen semen after cervical AI; (ii) avoiding costs and disease risks associated with animal transport in pedigree breeding for sire-referencing purposes; and (iii) helping to drive sheep genetic improvement programs by (a) making the use of AI in improvement programs more logistically achievable; (b) facilitating the use of AI in synchronized matings, (c) generating strong genetic linkages between flocks, (d) easing the high demand on rams caused by pedigree breeders breeding over the same 3-wk window (late July/early August), and (e) facilitating structured progeny testing schemes on commercial farms to feed back into the pedigree flocks, thus increasing the accuracy of selection. With this background, the objective of this study was to examine the effect of semen diluent, storage duration, and storage temperature on the viability and fertility of fresh ram semen. 2. Materials and methods All chemicals were obtained from Sigma (St. Louis, MO, USA) unless otherwise indicated. All semen samples were collected and assessed at the Teagasc Animal Production Research Centre, Athenry, Ireland. 2.1. Collection and preparation of semen Semen was collected from nine rams (Ovis aries) by artificial vagina. After collection, each ejaculate was assessed for volume, motility, wave motion, and concentration. Only ejaculates with a wave motion scoring > 3 on a scale of 0 to 5 and with a sperm concentration of more than 2.5 109/mL were accepted. All acceptable ejaculates were pooled and diluted to a final sperm concentration of 800 106/mL in each diluent. Depending on the experiment, semen was diluted in either a standard skim milk–egg yolk diluent or other commercially available diluents: OviPro (Minitub, Tiefenbach, Germany), AndroMed (Minitub), or INRA 96 (IMV Technologies, L’Aigle, France). Diluted semen was then allowed to adapt to room temperature for 30 min and then stored as described later. 2.2. Sperm motility and viability assessment A sample of diluted semen was removed to a prewarmed slide and covered with a coverslip. Motility was subjectively assessed using a phase-contrast microscope at 200 magnification and a 6-point scale, (0 = nonmotile to 5 = rapid motility). For assessment of
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viability, spermatozoa were stained as per the manufacturer’s guidelines using the Live/Dead Sperm Viability Kit (Molecular Probes, Eugene, OR, USA). Briefly, semen was diluted (1:30) in PBS, to which 5 mL SYBR 14 (final concentration 100 nM) had been added. This was vortexed and incubated for 10 min at 37 8C, following which 5 mL of propidium iodide (final concentration 12 mM) was added, and the solution was further incubated for 5 min at 37 8C. Two 5-mL aliquots were placed on a prewarmed slide; each aliquot was covered with a coverslip and assessed under a fluorescent microscope (Olympus BX 60, Dublin, Ireland). Five fields of view were assessed per aliquot. 2.3. Oocyte recovery and in vitro maturation The conditions for oocyte maturation were as described previously [19]. Briefly, ovaries from mature crossbred ewes were collected at a local abattoir and transported to the laboratory in phosphate-buffered saline (PBS) at 39 8C. Cumulus-oocyte complexes (COCs) were recovered by aspiration into sterile tubes containing 2 mL of aspiration medium (HEPESbuffered TCM-199 + 1.3% fetal calf serum + 0.45 mg/mL heparin and 0.05 mg/mL gentamicin). Cumulus-oocyte complexes were located under a stereomicroscope and washed through two dishes of aspiration medium followed by two dishes of maturation medium (TCM-199 + 10 ng/mL epidermal growth factor + 10% fetal calf serum + 200 mg/mL folliclestimulating hormone). Groups of COCs were then transferred into 4-well plates (approximately 50 oocytes per well) containing 500 mL maturation medium per well and incubated for 22 to 24 h in a humidified atmosphere of 5% CO2 in air at 39 8C. 2.4. In vitro fertilization Following in vitro maturation, mature COCs were placed in 0.5% hyaluronidase for 10 sec to remove excess cumulus cells and then washed in PBS and then in in vitro fertilization (IVF) medium (synthetic oviductal fluid [SOF] containing 1.3% fetal calf serum and 0.05 mg/mL gentamicin) before being transferred into 4-well dishes containing 250 mL SOF per well (25 oocytes per well). Preparation of sperm for IVF involved washing a 100-mL sample of fresh semen twice by centrifugation in 5 mL SOF at 100 g for 5 min. The resulting pellet was diluted in SOF to a final sperm concentration of 4 106/mL. A 250-mL aliquot of this sample was added to the oocytes to give a final sperm concentration of 2 106/mL. Dishes containing
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oocytes and spermatozoa were incubated for approximately 20 h at 39 8C in a humidified atmosphere of 5% CO2 in air. 2.5. In vitro culture At approximately 20 h postinsemination, presumptive zygotes were denuded of cumulus cells by vortexing in 3 mL PBS for 2 min. Zygotes were then washed three times in PBS and once in culture medium (SOF containing 6 mL/mL BME amino acids + 1 mL/ mL MEM amino acids + 0.05% mg/mL gentamicin) before being transferred in groups of approximately 25 into 25-mL droplets of medium under oil. Culture took place at 39 8C in an atmosphere of 5% CO2, 5% O2, and 90% N2. The proportion of oocytes undergoing cleavage was recorded at 48 h postinsemination. Blastocyst development was recorded daily from Day 6 to 9 postinsemination. 2.6. Experimental design 2.6.1. Experiment 1: Effect of duration of storage, storage temperature, and semen diluent on the motility and viability of fresh ram spermatozoa The aim of this experiment was to examine the effect of duration of storage, storage temperature, and semen diluent on the motility and viability of fresh ram sperm. Pooled fresh semen was diluted in one of four diluents, (1) standard skim milk [5], (2) AndroMed, (3) OviPro, or (4) INRA 96, and then stored in sealed 0.25-mL straws (Minitub) at either 5 8C or 15 8C. Motility and viability were assessed up to 72 h after semen collection as described earlier. 2.6.2. Experiment 2: Effect of duration of fresh semen storage and storage diluent on the ability of ram spermatozoa to penetrate artificial mucus The aim of this experiment was to assess the ability of semen stored for 6, 24, 48, or 72 h postcollection to penetrate artificial mucus (10 mg/mL sodium hyaluronate; MAP-5, Bioniche, Animal Health, Clonee, Ireland) using an assay modified from Gillan et al. [20]. Fresh ram semen was collected as above and diluted in one of three diluents (standard skim milk, AndroMed, or INRA 96) and stored at 5 8C. Artificial mucus consisting of 60% MAP 5 and 40% PBS was prepared [20]. A sample of semen was diluted to a final sperm concentration of 20 106/mL. To aid visibility, sperm were stained with Hoechst 33342 for 5 min at 37 8C. Flat capillary tubes (0.3 mm 3.0 mm 100 mm; Composite Metal Services, Shipley, West
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Yorkshire, UK) were marked at 10-mm intervals, loaded with artificial mucus, and placed vertically (three per diluent) in a 1.5-mL Eppendorf tube containing 250 mL of the stained sperm solution and incubated at 37 8C for 10 min. After incubation, the tubes were placed on a hot plate at 45 8C for 30 sec to kill and therefore immobilize the sperm. Sperm were counted from wall to wall across the tube at 10, 20, 30, 50, and 70 mm using a fluorescent microscope (400; Olympus BX 60). 2.6.3. Experiment 3: Effect of duration of fresh ram semen storage and storage diluent on development after IVF This experiment was performed to compare the ability of fresh ram spermatozoa stored for up to 72 h in standard skim milk, AndroMed, or INRA 96 at 5 8C to fertilize oocytes in vitro. In a preliminary experiment, storage of semen in INRA 96 at 15 8C resulted in a cleavage rate of <5% at 72 h postcollection. Therefore, in all subsequent experiments, semen storage was at 5 8C only. Thus, semen was collected from the same rams as used in Experiment 1, diluted, and stored in 15mL tubes at 5 8C. At 6, 24, and 72 h postcollection, diluted semen was used to inseminate in vitro–matured oocytes as described above. Oocyte collection was timed so that for each diluent, all semen storage times were evaluated for each semen collection (Table 1). The evaluation of fertilization ability was replicated with 17 batches (collections) of oocytes. For each replicate, matured oocytes were divided into equal groups, and each group was randomly inseminated with semen stored in one of the three diluents. 2.6.4. Experiment 4: Effect of duration of semen storage on fertility after cervical AI using fresh ram semen The aim of this experiment was to evaluate the in vivo fertility of fresh ram semen stored in INRA 96 at 5 8C for various periods prior to cervical AI. Multiparous ewes (n = 186) of various breeds (Scottish Blackface [n = 61], Suffolk [n = 13], Texel [n = 9], Cambridge
[n = 3], Belclare [n = 19], Belclare Scottish Blackface [n = 74], and Belclare Texel [n = 7]) were cervically inseminated to a synchronized estrus with fresh semen stored for 0, 24, 48, or 72 h. Synchronization was achieved via insertion of an intravaginal progesterone pessary (Cronolone, Intervet, Boxmeer, The Netherlands, 25 mg) for 12 d followed by equine chorionic gonadotropin (eCG; 400 IU) at pessary removal. Ewes were cervically inseminated 52 h after pessary removal by using an insemination pipette with a bent tip (Minitub). The experiment was replicated twice. For Replicate 1, pregnancy rate was determined by failure to return to estrus within 25 d of AI after exposure (from Day 14 after AI) to a panel of mature rams fitted with crayon markers. Pregnancy was terminated by administration of prostaglandin F2a, and after an interval of at least 8 d all ewes were re-synchronized and rerandomized to semen treatment for Replicate 2. Information on score for mucus secretion (0 = none to 3 = copious) evident at insemination and the depth to which it was possible to insert the insemination pipette into the cervical canal (0 = no penetration to 3 = deep penetration) were recorded at the time of AI. Ovulation rate and litter size were recorded after slaughter at about Day 25 postinsemination for the second replicate. 2.7. Statistical analysis The motility and viability (percentage live cells) for pooled semen samples were analyzed using Proc MIXED of SAS (SAS, Version 8, Cary, NC) in an initial model with fixed effects for temperature, time, diluent, and all two- and three-way interactions with replicate by diluent term treated as random. Because there were highly significant interactions involving storage temperature and the variances differed between the two temperatures, the data were finally analyzed separately for each temperature with fixed effects for time, diluent, and time by diluent interaction and replicate by diluent as a random term. Sperm mucus penetration was analyzed using Proc MIXED of SAS
Table 1 Experimental design for Experiment 3. Semen was collected once per week and used to inseminate in vitro–matured oocytes at 6, 24, or 72 h after semen collection following storage at 5 8C*. Monday
Tuesday
Wednesday
IVM Rep 1
Semen collection IVF Rep 1 (6 h) IVM Rep 2
IVF Rep 2 (24 h)
IVM, oocyte collection and in vitro maturation; IVF, oocyte in vitro fertilization; Rep, replicate. * Ovaries were only available from abattoirs on Monday, Tuesday, and Thursday each week.
Thursday
Friday
IVM Rep 3
IVF Rep 3 (72 h)
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following square-root transformation of the sperm count. The model had fixed effects for diluent, storage time, position along the capillary tube, and all two-way interactions, and replicate by time as a random term. In vitro fertility data for proportion of oocytes that cleaved, proportion that reached the blastocyst stage on Days 6, 7, 8, and 9, and the proportion of blastocysts that hatched were transformed (arcsine) prior to analysis using Proc MIXED of SAS and a model with fixed effects for treatment and storage time and their interaction, and a random term for oocyte batch was fitted. Differences among means were evaluated using the Tukey adjustment for multiple comparisons. In vivo fertility was analyzed using Proc GENMOD of SAS with a logit link function used to fit a model that included effects for treatment, ewe breed, and replicate. Because of the small number of Cambridge and Belclare Texel ewes and the fact that the Belclare and Cambridge ewes included carriers of major genes for ovulation rate [21], these classes were combined for analysis to form a group with a high natural ovulation rate. The initial model included two-way interaction terms, but these were dropped from the final model as none approached statistical significance.
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Table 2 Mean estimates of sperm motility and viability after storage of fresh ram sperm at 5 or 15 8C for 72 h postcollection (Experiment 1). Diluent
Temperature, 8C
Motility*
Viability, %
Skim milk AndroMed OviPro INRA 96 s.e. Skim milk AndroMed OviPro INRA 96 s.e.
5 5 5 5
2.3 3.2 3 2.5 0.21 0 1.16 0.16 0.16 0.23
68.0 62.7 58.2 55.7 2.91 31.8 35.7 26.8 31.3 3.47
15 15 15 15
s.e. = standard error. * Motility was subjectively assessed using a phase-contrast microscope at 200 magnification and a 6-point scale (0 = nonmotile to 5 = rapid motility).
analyses of the data for each storage temperature. When sperm were stored at 15 8C, there was a significant diluent by time interaction for viability. This effect was not significant when storage was at 5 8C. The pattern of results for motility was similar; there was a significant time by diluent interaction when sperm were stored at 15 8C, but this effect was not significant at 5 8C.
3. Results
3.2. Experiment 2
3.1. Experiment 1
There was a significant diluents by time interaction. The number of spermatozoa penetrating mucus differed among diluents at 6 and 24 h postcollection, but no differences were evident at 48 or 72 h (Fig. 2). Among the diluents tested, the number of spermatozoa stored in INRA 96 penetrating the mucus remained relatively constant across all time points.
The residual correlation between the proportion of live sperm (viability) in duplicate aliquots was 0.28; the mean value was used in the final analysis. Storage temperature had a highly significant effect on both motility and viability; at 5 8C, viability remained relatively stable between 24 and 72 h postcollection, whereas at 15 8C, viability declined in a linear fashion as time of storage increased (Fig. 1). The estimates for motility score and viability percentage at 72 h postcollection are presented in Table 2 based on separate
Fig. 1. Proportion of live sperm up to 72 h after semen collection following semen storage at 5 8C (—) or 15 8C (– –) in skim milk (^), OviPro (&), AndroMed (~), or INRA 96 (*) (Experiment 1).
3.3. Experiment 3 Results of the preliminary experiment are shown in Table 3. The proportion of oocytes undergoing cleavage
Fig. 2. Least square means for the effect of storage diluent on the number of fresh ram spermatozoa penetrating artificial mucus at 6, 24, 48, and 72 h postcollection (Experiment 2).
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Table 3 Cleavage and development to the blastocyst stage after IVF with fresh ram sperm stored in INRA 96 for 6, 24, or 72 h at 15 8C (Experiment 3). Time, h
Number of oocytes
Cleavage, % (95% confidence interval)
Blastocyst rate, % (95% confidence interval)
6 24 72
96 107 124
74.5 (61.2–85.8) 83.5 (71.7–92.6) 2.6 (0.0–9.0)
20.9 (9.8–34.7) 27.2 (14.7–41.9) 1.0 (0.3–6.3)
Table 4 Cleavage and blastocyst development after IVF with fresh ram sperm stored at 5 8C for 6, 24, or 72 h postcollection in one of three diluents. Time, h
Diluent
Number of oocytes
Cleavage rate, % (95% confidence interval)
Blastocyst rate, % (95% confidence interval)
6
Skim milk AndroMed INRA 96 Skim milk AndroMed INRA 96 Skim milk AndroMed INRA 96
195 181 178 321 318 332 291 268 282
32.7 42.7 37.1 63.5 71.6 63.3 59.1 60.6 62.7
10.5 (3.1–21.8) 9.6 (2.5–20.5) 9.2 (2.3–20.0) 25.2 (13.2–39.7) 34.7 (20.9–50.0) 19.7 (9.0–33.3) 22.1 (11.6–34.8) 15.0 (6.4–26.4) 21.7 (11.3–34.4)
24
72
(10.8–59.7) (18.0–69.6) (13.8–64.2) (38.8–85.0) (47.4–90.6) (38.6–84.8) (34.4–81.5) (35.8–82.7) (37.9–84.3)
after IVF and the proportion developing to the blastocyst stage are presented in Table 4. After storage at 5 8C, cleavage rate was unaffected by the duration of storage (P = 0.2) or diluent (P = 0.3), and there was no evidence for an interaction (P = 0.8). In the case of blastocyst rate, there was some evidence for an effect of diluent (P = 0.08) and a significant time by diluent interaction reflecting the numerical superiority of AndroMed at the 24-h time point, whereas this diluent was poorest at 72 h, with little difference among the diluents at the 6-h time point. 3.4. Experiment 4 Sperm motility was lower at 48 and 72 h compared with that for fresh (0 h) and at 24 h, consistent with the
Fig. 3. Pregnancy rate in ewes after cervical AI using fresh ram semen stored for 0, 24, 48, or 72 h (vertical bars represent the 95% confidence interval for the mean) (Experiment 4). The estimated means (95% confidence interval) for pregnancy rate (%) were 60.2 (46.3 to 72.7), 52.2 (39.9 to 64.1), 30.4 (20.1 to 42.1), and 18.3 (10.9 to 29.4) for 0, 24, 48, and 72 h, respectively.
results of Experiment 1 for semen stored in INRA 96 at 5 8C (data not shown). Pregnancy rate, adjusted for effects of breed and replicate, are presented in Fig. 3. Pregnancy rate decreased up to 72 h after semen collection (P < 0.001). The decline was essentially linear (P < 0.001) as the quadratic term did not approach significance. The effects of ewe breed and replicate were also significant. The estimated breed means (95% confidence interval) for pregnancy rate (%) were 25.5 (10.0 to 51.1), 34.1 (17.0 to 56.6), 64.2 (54.2 to 73.1), 50.0 (40.4 to 58.9), and 35.6 (23.0 to 50.7) for Texel, Suffolk, Scottish Blackface, Belclare Scottish Blackface, and High ovulation rate groups, respectively. There was no evidence for any breed by treatment or replicate by treatment interactions. A total of 76 ewes yielded litter size data, and ovulation rate information was available for 169 ewes. There was no significant association between treatment and ovulation rate assessed postmortem (Table 5). Also, there was no evidence that treatment affected the litter size of ewes that conceived to AI. Information on score for mucus secretion (0 = none to 3 = copious) evident at insemination and the depth to which it was possible to insert the insemination pipette into the cervical canal (penetration score, 0 = no penetration to 3 = deep penetration) were recorded at the time of AI. The distribution of these scores is shown in Fig. 4. The analyses of the scores is summarized in Table 6. There was no evidence for any effect of breed or treatment on either mucus or penetration scores. The repeatability estimates for mucus and penetration scores
L. O’Hara et al. / Theriogenology 73 (2010) 541–549 Table 5 Ovulation rate and litter size for ewes in AI study (Experiment 4). Factor
Ovulation rate*
Litter size
Treatment (time, h) 0 24 48 72 F-test
2.37 0.179 2.21 0.158 2.49 0.162 2.07 0.176 NS
1.79 0.204 1.68 0.206 1.77 0.251 1.45 0.338 NS
Ewe breed Texel Suffolk Scottish Blackface Belclare Scottish Blackface High ovulation rate group F-test
1.79 0.294 2.07 0.263 1.72 0.112 2.28 0.109 3.44 0.176 P < 0.001
1.48 0.362 1.07 0.415 1.56 0.132 1.68 0.142 2.21 0.235 P = 0.109
NS, not significant. * Adjusted for pregnancy status.
were 0.23 and 0.22, respectively (P < 0.01). There was no evidence (P = 0.6) for any association between penetration score and pregnancy rate, but a positive association between pregnancy rate and mucus score approached formal significance (P = 0.08; Proc GENMOD with mucus score as a covariate). 4. Discussion This study involved the evaluation of the effect of storage of fresh ram semen for up to 72 h postcollection in terms of sperm motility and viability as well as more functional measures including the ability of sperm to penetrate artificial mucus, to fertilize oocytes in vitro, and ultimately to establish pregnancy in ewes after cervical insemination. The main findings are (1) fresh storage of ram semen at 5 8C is superior to storage at 15 8C, (2) a high proportion of sperm retain their ability to penetrate artificial mucus for up to 72 h, (3) storage at 5 8C for 72 h postcollection does not diminish the ability of sperm to fertilize oocytes in vitro, and (4)
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increased duration of storage is associated with a linear decrease in pregnancy rate in ewes after AI. The liquid storage of ram semen has been extensively reviewed in recent years [13,22]. Irrespective of the diluent used, dilution rate, temperature, or conditions of storage, the quality of spermatozoa deteriorates as the duration of storage increases. The main changes that occur during storage include a reduction in motility, morphologic integrity, and fertility of spermatozoa. It is hypothesized that these changes are accompanied by a decline in the survival of spermatozoa in the female reproductive tract and may be associated with an increase in embryonic loss. Although several groups have assessed the effect of storage on motility and viability [2,7], relatively few have translated the in vitro observations to field fertility [23]. This is important because there are a plethora of studies in the literature examining the ability to predict field fertility from laboratory tests in both sheep [19,24] and cattle [25,26]. Most of these results are equivocal. Traditional in vitro evaluation of semen quality involves the subjective assessment of motility and the proportion of sperm with normal morphology. Other, more elaborate tests include measures of viability, acrosomal integrity, objective computer-based assessment of motility, and IVF. Whereas some success has been achieved using these measures, few, if any, single in vitro tests show a reliable and repeatable correlation with field fertility. In Experiment 1, storage temperature had a significant effect on sperm motility and viability up to 72 h postcollection, irrespective of diluent; storage at 15 8C was detrimental to both parameters, whereas at 5 8C both motility and viability remained relatively constant. Although motility is an important measure of sperm functionality, it is difficult to relate this parameter to field fertility. Sperm can retain their motility in vitro for a relatively long period of time. For example, LopezSaez et al. [2] examined the effect of three diluents
Table 6 Ewe breed effects on scores from mucus and penetration at AI (Experiment 4).
Fig. 4. Frequency distribution for mucus and depth of penetration scores at the time of cervical insemination (Experiment 4).
Ewe breed
Penetration score
Mucus score
Suffolk Texel Scottish Blackface Belclare Scottish Blackface High ovulation rate group F-test
1.8 0.17 1.4 0.20 1.4 0.08 1.5 0.07 1.4 0.12 NS
2.2 0.09 1.9 0.10 2.0 0.04 2.1 0.04 2.0 0.06 NS
NS, not significant.
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(skim milk and two Tris-based extenders) on ram semen quality in terms of motility and membrane integrity after storage at 5 8C for up to 16 d. Whereas the percentage motile sperm decreased with time, a significant proportion (>40%) was still motile after 16 d storage in the two Tris-based diluents. Similarly, a proportion of sperm were capable of fertilizing oocytes in vitro and in vivo after storage at 5 8C for 7 d and even 14 d [27,28]. The current findings that storage at 5 8C was associated with only minor changes in motility and viability up to 72 h compared with the major decline when storage was at 15 8C are consistent with those of Paulenz et al. [7] who reported similar results for motility, along with acrosome integrity and capacitation status after 0 to 30 h storage of liquid ram semen at 5 8C compared with storage at 20 8C. The ability of sperm to move through the mucus-rich environment of the female reproductive tract, particularly the cervix, was assayed by assessing sperm migration in artificial mucus [20]. In comparison with cervical mucus, artificial mucus provides a standard medium with no biological variability allowing standardized assessment of migration. Gillan et al. [20] showed that spermatozoa from bulls with high fertility migrate through such mucus in larger proportions and that the vanguard spermatozoon migrated a greater distance than spermatozoa from low-fertility bulls. The number of spermatozoa penetrating mucus in the current study differed slightly among diluents at 6 and 24 h postcollection but was relatively constant at 48 and 72 h. Among the diluents tested, the number of spermatozoa stored in INRA 96 penetrating the mucus remained relatively constant across all time points. It has been stated that one of the constraining factors in the use of fresh stored ram semen at 15 8C is that the sperm fertilizing ability declines with time (6 to 12 h) [2]. Our results would contradict this notion somewhat; whereas storage for 72 h at 15 8C resulted in a dramatic reduction in the proportion of oocytes undergoing cleavage and, as a consequence, developing to the blastocyst stage, the fertilizing ability of the sperm was unaffected up to 24 h postcollection. Furthermore, irrespective of diluent used, neither cleavage rate nor blastocyst development decreased up to 72 h postcollection when storage was at 5 8C (Table 4). Maxwell and Stojanov [27] reported that a proportion of sperm were capable of fertilizing oocytes in vitro after storage at 5 8C for 7 d and even 14 days. The increase in oocyte cleavage after IVF and blastocyst development between 6 and 24 h of storage in the current study is probably a reflection of the suboptimal or incomplete capacitation status of the sperm 6 h after collection.
Successful fertilization in vivo depends not only on the performance of the sperm but also on the transport of sperm and site of deposition in the female reproductive tract. After confirming that fresh ram semen stored at 5 8C, irrespective of diluent, was capable of fertilizing oocytes up to 72 h postcollection, the fertilizing ability of such semen in vivo was examined. In studies reviewed by Maxwell and Salamon [22], fertility declined rapidly when semen stored for more than 24 h was used for cervical insemination (by 10% to 35% per day of storage); lambing rates for semen stored for 24, 48, and 72 h were 45% to 50%, 25% to 30%, and 15% to 20%, respectively. It is known that the cervix represents a formidable obstacle that sperm must traverse after natural mating or cervical insemination. With compromised sperm (i.e., frozen-thawed [14] or chilled-stored [28]), pregnancy rates are higher after intrauterine insemination compared with those after cervical insemination suggesting that issues related to sperm transport in the female reproductive tract play a significant role in the decline in fertility seen with such spermatozoa. Salamon et al. [28] showed that the fertilizing capacity of ram spermatozoa is retained for up to 10 d of storage at 5 8C, but lambing rates after cervical insemination declined substantially when the semen was stored for more than 3 d. In the study of Maxwell and Stojanov [27], the in vivo fertilization and lambing rates obtained after 7 d storage of semen in the presence of antioxidants were not higher than those reported by Salamon et al. [28] after 8 d, but the storage time for which the spermatozoa retained their fertilizing capacity (14 d) was beyond previous limits. Furthermore, pregnancies were obtained after intrauterine insemination with semen chilled-stored for 14 d. In the current study, semen stored in INRA 96 at 5 8C gave acceptable pregnancy rates after insemination up to 24 h postcollection (63% and 54% for 0 and 24 h, respectively). Pregnancy rates decreased after 24 h, with rates of 30% and 20% for 48 and 72 h stored semen, respectively. These results are similar to those reviewed by Maxwell and Salamon [22]. Ewe breed had a significant effect on pregnancy rate. This effect was unexpected, as generally ewe breed has not been reported as a source of variation in conception rate after AI with fresh semen. It has, however, been a factor in the success of conception rate after the use of frozen-thawed semen in AI [4,5]. In conclusion, the results of this study indicate that ram semen cooled and stored at 15 8C has a shorter life span than that cooled to 5 8C. Furthermore, the commercially available diluents tested in this study did not differ significantly in terms of fresh ram semen
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performance in vitro when stored at 5 8C. Sperm storage in INRA 96 at 5 8C gave acceptable pregnancy rates up to 24 h after semen collection, and given that it can be used directly off the shelf, it could offer an alternative to the standard skim milk currently used. Where possible, cervical insemination should be performed within 24 h of semen collection to obtain acceptable pregnancy rates. In situations where this is not possible, semen stored at 5 8C can still be used for cervical insemination up to 3 d postcollection, although pregnancy rates will be decreased. Acknowledgments We acknowledge the technical staff at Teagasc Research Centre, Athenry, Ireland, for management of rams and collection of semen. We would also like to acknowledge Mary Wade at Lyons Research Farm, UCD, Newcastle, Ireland, for media preparation. This work was funded by Teagasc under the Walsh Fellowship Scheme. References [1] Fair S, Hanrahan JP, Donovan A, Duffy P, O’Meara CM, Lonergan P, Evans AC. Hormonal relationships during the periovulatory period among ewe breeds known to differ in fertility after cervical artificial insemination with frozen thawed semen. Anim Reprod Sci 2007;97:284–94. [2] Lopez-Saez A, Ortiz N, Gallego L, Garde JJ. Liquid storage (5 degrees C) of ram semen in different diluents. Arch Androl 2000;44:155–64. [3] Vishwanath R, Shannon P. Storage of bovine semen in liquid and frozen state. Anim Reprod Sci 2000;62:23–53. [4] Donovan A, Hanrahan JP, Lally T, Boland MP, Byrne GP, Duffy P, et al. AI for Sheep Using Frozen-Thawed Semen. End of Project Report: Sheep Series No. 11. Teagasc, Agriculture and Food Development Authority, 2001. [5] Donovan A, Hanrahan JP, Kummen E, Duffy P, Boland MP. Fertility in the ewe following cervical insemination with fresh or frozen-thawed semen at a natural or synchronised oestrus. Anim Reprod Sci 2004;84:359–68. [6] Eppleston J, Maxwell WMC. Recent attempts to improve the fertility of frozen ram semen inseminated into the cervix. Wool Technology and Sheep Breeding 1993;41:291–302. [7] Paulenz H, Soderquist L, Perez-Pe R, Berg KA. Effect of different extenders and storage temperatures on sperm viability of liquid ram semen. Theriogenology 2002;57:823–36. [8] Bailey JL, Bilodeau JF, Cormier N. Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. J Androl 2000;21:1–7. [9] Byrne GP, Lonergan P, Wade M, Duffy P, Donovan A, Hanrahan JP, Boland MP. Effect of freezing rate of ram spermatozoa on subsequent fertility in vivo and in vitro. Anim Reprod Sci 2000;62:265–75. [10] Maxwell WMC, Watson PF. Recent progress in the preservation of ram semen. Anim Reprod Sci 1996;42:55–65.
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[11] Salamon S, Maxwell WMC. Frozen storage of ram semen. 1. Processing, freezing, thawing and fertility after cervical insemination. Anim Reprod Sci 1995;37:185–249. [12] Salamon S, Maxwell WMC. Frozen storage of ram semen. 2. Causes of low fertility after cervical insemination and methods of improvement. Anim Reprod Sci 1995;38:1–36. [13] Salamon S, Maxwell WM. Storage of ram semen. Anim Reprod Sci 2000;62:77–111. [14] Fair S, Hanrahan JP, O’Meara CM, Duffy P, Rizos D, Wade M, et al. Differences between Belclare and Suffolk ewes in fertilization rate, embryo quality and accessory sperm number after cervical or laparoscopic artificial insemination. Theriogenology 2005;63:1995–2005. [15] Gil J, Lundeheim N, Soderquist L, Rodriguez-Martinez H. Influence of extender, temperature, and addition of glycerol on post-thaw sperm parameters in ram semen. Theriogenology 2003;59:1241–55. [16] Gil J, Rodriguez-Irazoqui M, Lundeheim N, Soderquist L, Rodriguez-Martinez H. Fertility of ram semen frozen in Bioexcell (R) and used for cervical artificial insemination. Theriogenology 2003;59:1157–70. [17] Marco-Jimenez F, Puchades S, Moce E, Viudes-De-Cartro MP, Vicente JS, Rodriguez M. Use of powdered egg yolk vs fresh egg yolk for the cryopreservation of ovine semen. Reprod Domest Anim 2004;39:438–41. [18] Matsuoka T, Imai H, Kohno H, Fukui Y. Effects of bovine serum albumin and trehalose in semen diluents for improvement of frozen-thawed ram spermatozoa. J Reprod Dev 2006;52:675–83. [19] O’Meara CM, Hanrahan JP, Donovan A, Fair S, Rizos D, Wade M, et al. Relationship between in vitro fertilisation of ewe oocytes and the fertility of ewes following cervical artificial insemination with frozen-thawed ram semen. Theriogenology 2005;64:1797–808. [20] Gillan L, Kroetsch T, Maxwell WM, Evans G. Assessment of in vitro sperm characteristics in relation to fertility in dairy bulls. Anim Reprod Sci 2008;103:201–14. [21] Hanrahan JP, Gregan SM, Mulsant P, Mullen M, Davis GH, Powell R, Galloway SM. Mutations in the genes for oocytederived growth factors GDF9 and BMP15 are associated with both increased ovulation rate and sterility in Cambridge and Belclare sheep (Ovis aries). Biol Reprod 2004;70:900–9. [22] Maxwell WM, Salamon S. Liquid storage of ram semen: a review. Reprod Fertil Dev 1993;5:613–38. [23] Mara L, Accardo C, Pilichi S, Dattena M, Chessa F, Chessa B, et al. Benefits of TEMPOL on ram semen motility and in vitro fertility: a preliminary study. Theriogenology 2005;63:2243–53. [24] O’Meara CM, Hanrahan JP, Prathalingham NS, Owen JS, Donovan A, Fair S, et al. Relationship between in vitro sperm functional tests and in vivo fertility of rams following cervical artificial insemination of ewes with frozen-thawed semen. Theriogenology 2008;69:513–22. [25] Januskauskas A, Johannisson A, Rodriguez-Martinez H. Subtle membrane changes in cryopreserved bull semen in relation with sperm viability, chromatin structure, and field fertility. Theriogenology 2003;60:743–58. [26] Larsson B, Rodriguez-Martinez H. Can we use in vitro fertilization tests to predict semen fertility? Anim Reprod Sci 2000;60– 61:327–36. [27] Maxwell WM, Stojanov T. Liquid storage of ram semen in the absence or presence of some antioxidants. Reprod Fertil Dev 1996;8:1013–20. [28] Salamon S, Maxwell WMC, Firth JH. Fertility of ram semen after storage at 5 8C. Anim Reprod Sci 1979;2:373–85.
Theriogenology 73 (2010) 541–549 www.theriojournal.com
Effect of storage duration, storage temperature, and diluent on the viability and fertility of fresh ram sperm L. O’Hara a,c, J.P. Hanrahan a, L. Richardson a,c, A. Donovan a, S. Fair b, A.C.O. Evans c, P. Lonergan c,* a Teagasc, Animal Production Research Centre, Athenry, Ireland Physiology, School of Medicine, National University of Ireland Galway, Galway, Ireland c School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Ireland b
Received 20 August 2009; received in revised form 3 October 2009; accepted 6 October 2009
Abstract Cervical artificial insemination (AI) in sheep with fresh semen yields a much higher pregnancy rate than when frozen-thawed semen is used, and consequently frozen semen is only acceptable for laparoscopic insemination. The short life span of fresh semen is a major constraint on the use of AI in genetic improvement programs for sheep. The main objective of this study was to examine the effects of storage conditions on viability and fertilization ability of fresh ram (Ovis aries) semen up to 72 h postcollection. Experiment 1 was designed to evaluate the effect of diluent type (standard skim milk, AndroMed, OviPro, and INRA 96) and storage temperature (5 8C and 15 8C) on the motility and viability of fresh ram semen. Storage temperature, irrespective of diluent, had a significant effect on both motility and viability. Storage at 5 8C maintained acceptable motility and viability up to 72 h compared with that of storage at 15 8C. In Experiment 2, the penetrating ability of fresh ram semen, diluted in either skim milk, AndroMed, or INRA 96, was assessed using artificial mucus. Flat capillary tubes containing artificial mucus were suspended in 250 mL semen at a sperm concentration of 20 106/ mL. Semen was stored at 5 8C and tested after 6, 24, 48, and 72 h. There was a significant diluent by time interaction. In Experiment 3, the fertilizing ability of fresh ram semen stored at 5 8C was evaluated in vitro. Fresh semen (diluted in either skim milk, AndroMed, or INRA 96) was added to matured ewe oocytes at 6, 24, or 72 h after semen collection. Cleavage rate was recorded at 48 h postinsemination, and blastocyst development was recorded on Days 6 to 9. There was a significant treatment effect on cleavage and blastocyst rates; insemination of semen stored for 24 h resulted in higher rates than those for storage at 72 h. In Experiment 4, the fertilizing ability of fresh ram semen was evaluated in vivo. Semen was diluted in INRA 96, stored at 5 8C, and used to inseminate ewes on the day of collection or at 24, 48, and 72 h postcollection. Multiparous ewes were cervically inseminated at a synchronized estrus. Fertility rate decreased linearly (P < 0.001) up to 72 h after semen collection. # 2010 Elsevier Inc. All rights reserved. Keywords: AI; IVF; Semen extender; Sheep
1. Introduction Artificial insemination (AI), when used in conjunction with accurate progeny testing schemes, can * Corresponding author. Tel.: +353 1 6012147; fax: +353 1 6288421. E-mail address: [email protected] (P. Lonergan). 0093-691X/$ – see front matter # 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.10.009
substantially increase the rate of genetic progress compared with that of natural service and is the method of choice for dissemination of genetic improvement. Artificial insemination is an essential and wellestablished facilitator of genetic improvement in cattle, but the absence of an equally effective technology in sheep, where it would lead to increased accuracy of across-flock evaluation and higher selection intensity,
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represents a significant constraint for genetic improvement programs [1]. In sheep, whereas cervical AI with fresh semen yields an acceptable pregnancy rate, the short shelf life (10 to 14 h) of fresh semen [2] coupled with the natural limitation on the number of semen doses that can be obtained per ram per unit time plus small flock sizes seriously restricts the widespread use of individual sires. Several extenders have been developed for fresh storage of semen in cattle but, to date, none is capable of storing sperm for more than 2 to 3 d without a drop in fertility [3]; the situation is much worse in sheep. The widespread use of AI and the realization of its full potential depend on the use of frozen-thawed semen [4]. However, AI with frozen-thawed ram semen has not been widely adopted for sheep mainly due to the very poor fertility obtained after cervical insemination compared with the pregnancy rate after cervical insemination with fresh semen or that obtained with laparoscopic intrauterine insemination [4–7]. The low fertility from cervical insemination with frozen-thawed semen is attributed to irreversible damage caused to the cells during the freeze-thaw process [8–12]. Of the high proportion (40% to 60%) of ram spermatozoa that maintain their motility after freeze thawing, only 20% to 30% may remain biologically undamaged and therefore capable of successful fertilization; the few remaining undamaged spermatozoa are then often unable to transverse the cervical barrier and female reproductive tract to the site of fertilization (reviewed by Salamon and Maxwell [13]). Evidence from our group would support this notion; thus, we have shown that pregnancy rate after laparoscopic AI using frozenthawed semen is higher than that after cervical AI [14] and in addition that the number of accessory sperm on the zona pellucida is significantly higher than that after cervical AI, suggesting a problem with sperm transport to the site of fertilization [14]. However, laparoscopic AI is expensive, requires technical expertise, and raises concerns for animal welfare and thus is not likely to be a viable long-term alternative to the development of strategies for improving pregnancy rate to cervical AI. Storage diluents and techniques have been developed and adapted with the aim of improving the cryopreservation of ram semen [15–18], but fertility after cervical AI with frozen-thawed sperm still remains significantly lower than that achieved with fresh semen. Therefore, cervical AI with fresh semen would be a more attractive option especially if the effective length of fresh semen could be extended to 48 h. This would represent a major improvement on current practice and would have several advantages including (i) bypassing
problems of poor fertility associated with frozen semen after cervical AI; (ii) avoiding costs and disease risks associated with animal transport in pedigree breeding for sire-referencing purposes; and (iii) helping to drive sheep genetic improvement programs by (a) making the use of AI in improvement programs more logistically achievable; (b) facilitating the use of AI in synchronized matings, (c) generating strong genetic linkages between flocks, (d) easing the high demand on rams caused by pedigree breeders breeding over the same 3-wk window (late July/early August), and (e) facilitating structured progeny testing schemes on commercial farms to feed back into the pedigree flocks, thus increasing the accuracy of selection. With this background, the objective of this study was to examine the effect of semen diluent, storage duration, and storage temperature on the viability and fertility of fresh ram semen. 2. Materials and methods All chemicals were obtained from Sigma (St. Louis, MO, USA) unless otherwise indicated. All semen samples were collected and assessed at the Teagasc Animal Production Research Centre, Athenry, Ireland. 2.1. Collection and preparation of semen Semen was collected from nine rams (Ovis aries) by artificial vagina. After collection, each ejaculate was assessed for volume, motility, wave motion, and concentration. Only ejaculates with a wave motion scoring > 3 on a scale of 0 to 5 and with a sperm concentration of more than 2.5 109/mL were accepted. All acceptable ejaculates were pooled and diluted to a final sperm concentration of 800 106/mL in each diluent. Depending on the experiment, semen was diluted in either a standard skim milk–egg yolk diluent or other commercially available diluents: OviPro (Minitub, Tiefenbach, Germany), AndroMed (Minitub), or INRA 96 (IMV Technologies, L’Aigle, France). Diluted semen was then allowed to adapt to room temperature for 30 min and then stored as described later. 2.2. Sperm motility and viability assessment A sample of diluted semen was removed to a prewarmed slide and covered with a coverslip. Motility was subjectively assessed using a phase-contrast microscope at 200 magnification and a 6-point scale, (0 = nonmotile to 5 = rapid motility). For assessment of
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viability, spermatozoa were stained as per the manufacturer’s guidelines using the Live/Dead Sperm Viability Kit (Molecular Probes, Eugene, OR, USA). Briefly, semen was diluted (1:30) in PBS, to which 5 mL SYBR 14 (final concentration 100 nM) had been added. This was vortexed and incubated for 10 min at 37 8C, following which 5 mL of propidium iodide (final concentration 12 mM) was added, and the solution was further incubated for 5 min at 37 8C. Two 5-mL aliquots were placed on a prewarmed slide; each aliquot was covered with a coverslip and assessed under a fluorescent microscope (Olympus BX 60, Dublin, Ireland). Five fields of view were assessed per aliquot. 2.3. Oocyte recovery and in vitro maturation The conditions for oocyte maturation were as described previously [19]. Briefly, ovaries from mature crossbred ewes were collected at a local abattoir and transported to the laboratory in phosphate-buffered saline (PBS) at 39 8C. Cumulus-oocyte complexes (COCs) were recovered by aspiration into sterile tubes containing 2 mL of aspiration medium (HEPESbuffered TCM-199 + 1.3% fetal calf serum + 0.45 mg/mL heparin and 0.05 mg/mL gentamicin). Cumulus-oocyte complexes were located under a stereomicroscope and washed through two dishes of aspiration medium followed by two dishes of maturation medium (TCM-199 + 10 ng/mL epidermal growth factor + 10% fetal calf serum + 200 mg/mL folliclestimulating hormone). Groups of COCs were then transferred into 4-well plates (approximately 50 oocytes per well) containing 500 mL maturation medium per well and incubated for 22 to 24 h in a humidified atmosphere of 5% CO2 in air at 39 8C. 2.4. In vitro fertilization Following in vitro maturation, mature COCs were placed in 0.5% hyaluronidase for 10 sec to remove excess cumulus cells and then washed in PBS and then in in vitro fertilization (IVF) medium (synthetic oviductal fluid [SOF] containing 1.3% fetal calf serum and 0.05 mg/mL gentamicin) before being transferred into 4-well dishes containing 250 mL SOF per well (25 oocytes per well). Preparation of sperm for IVF involved washing a 100-mL sample of fresh semen twice by centrifugation in 5 mL SOF at 100 g for 5 min. The resulting pellet was diluted in SOF to a final sperm concentration of 4 106/mL. A 250-mL aliquot of this sample was added to the oocytes to give a final sperm concentration of 2 106/mL. Dishes containing
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oocytes and spermatozoa were incubated for approximately 20 h at 39 8C in a humidified atmosphere of 5% CO2 in air. 2.5. In vitro culture At approximately 20 h postinsemination, presumptive zygotes were denuded of cumulus cells by vortexing in 3 mL PBS for 2 min. Zygotes were then washed three times in PBS and once in culture medium (SOF containing 6 mL/mL BME amino acids + 1 mL/ mL MEM amino acids + 0.05% mg/mL gentamicin) before being transferred in groups of approximately 25 into 25-mL droplets of medium under oil. Culture took place at 39 8C in an atmosphere of 5% CO2, 5% O2, and 90% N2. The proportion of oocytes undergoing cleavage was recorded at 48 h postinsemination. Blastocyst development was recorded daily from Day 6 to 9 postinsemination. 2.6. Experimental design 2.6.1. Experiment 1: Effect of duration of storage, storage temperature, and semen diluent on the motility and viability of fresh ram spermatozoa The aim of this experiment was to examine the effect of duration of storage, storage temperature, and semen diluent on the motility and viability of fresh ram sperm. Pooled fresh semen was diluted in one of four diluents, (1) standard skim milk [5], (2) AndroMed, (3) OviPro, or (4) INRA 96, and then stored in sealed 0.25-mL straws (Minitub) at either 5 8C or 15 8C. Motility and viability were assessed up to 72 h after semen collection as described earlier. 2.6.2. Experiment 2: Effect of duration of fresh semen storage and storage diluent on the ability of ram spermatozoa to penetrate artificial mucus The aim of this experiment was to assess the ability of semen stored for 6, 24, 48, or 72 h postcollection to penetrate artificial mucus (10 mg/mL sodium hyaluronate; MAP-5, Bioniche, Animal Health, Clonee, Ireland) using an assay modified from Gillan et al. [20]. Fresh ram semen was collected as above and diluted in one of three diluents (standard skim milk, AndroMed, or INRA 96) and stored at 5 8C. Artificial mucus consisting of 60% MAP 5 and 40% PBS was prepared [20]. A sample of semen was diluted to a final sperm concentration of 20 106/mL. To aid visibility, sperm were stained with Hoechst 33342 for 5 min at 37 8C. Flat capillary tubes (0.3 mm 3.0 mm 100 mm; Composite Metal Services, Shipley, West
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Yorkshire, UK) were marked at 10-mm intervals, loaded with artificial mucus, and placed vertically (three per diluent) in a 1.5-mL Eppendorf tube containing 250 mL of the stained sperm solution and incubated at 37 8C for 10 min. After incubation, the tubes were placed on a hot plate at 45 8C for 30 sec to kill and therefore immobilize the sperm. Sperm were counted from wall to wall across the tube at 10, 20, 30, 50, and 70 mm using a fluorescent microscope (400; Olympus BX 60). 2.6.3. Experiment 3: Effect of duration of fresh ram semen storage and storage diluent on development after IVF This experiment was performed to compare the ability of fresh ram spermatozoa stored for up to 72 h in standard skim milk, AndroMed, or INRA 96 at 5 8C to fertilize oocytes in vitro. In a preliminary experiment, storage of semen in INRA 96 at 15 8C resulted in a cleavage rate of <5% at 72 h postcollection. Therefore, in all subsequent experiments, semen storage was at 5 8C only. Thus, semen was collected from the same rams as used in Experiment 1, diluted, and stored in 15mL tubes at 5 8C. At 6, 24, and 72 h postcollection, diluted semen was used to inseminate in vitro–matured oocytes as described above. Oocyte collection was timed so that for each diluent, all semen storage times were evaluated for each semen collection (Table 1). The evaluation of fertilization ability was replicated with 17 batches (collections) of oocytes. For each replicate, matured oocytes were divided into equal groups, and each group was randomly inseminated with semen stored in one of the three diluents. 2.6.4. Experiment 4: Effect of duration of semen storage on fertility after cervical AI using fresh ram semen The aim of this experiment was to evaluate the in vivo fertility of fresh ram semen stored in INRA 96 at 5 8C for various periods prior to cervical AI. Multiparous ewes (n = 186) of various breeds (Scottish Blackface [n = 61], Suffolk [n = 13], Texel [n = 9], Cambridge
[n = 3], Belclare [n = 19], Belclare Scottish Blackface [n = 74], and Belclare Texel [n = 7]) were cervically inseminated to a synchronized estrus with fresh semen stored for 0, 24, 48, or 72 h. Synchronization was achieved via insertion of an intravaginal progesterone pessary (Cronolone, Intervet, Boxmeer, The Netherlands, 25 mg) for 12 d followed by equine chorionic gonadotropin (eCG; 400 IU) at pessary removal. Ewes were cervically inseminated 52 h after pessary removal by using an insemination pipette with a bent tip (Minitub). The experiment was replicated twice. For Replicate 1, pregnancy rate was determined by failure to return to estrus within 25 d of AI after exposure (from Day 14 after AI) to a panel of mature rams fitted with crayon markers. Pregnancy was terminated by administration of prostaglandin F2a, and after an interval of at least 8 d all ewes were re-synchronized and rerandomized to semen treatment for Replicate 2. Information on score for mucus secretion (0 = none to 3 = copious) evident at insemination and the depth to which it was possible to insert the insemination pipette into the cervical canal (0 = no penetration to 3 = deep penetration) were recorded at the time of AI. Ovulation rate and litter size were recorded after slaughter at about Day 25 postinsemination for the second replicate. 2.7. Statistical analysis The motility and viability (percentage live cells) for pooled semen samples were analyzed using Proc MIXED of SAS (SAS, Version 8, Cary, NC) in an initial model with fixed effects for temperature, time, diluent, and all two- and three-way interactions with replicate by diluent term treated as random. Because there were highly significant interactions involving storage temperature and the variances differed between the two temperatures, the data were finally analyzed separately for each temperature with fixed effects for time, diluent, and time by diluent interaction and replicate by diluent as a random term. Sperm mucus penetration was analyzed using Proc MIXED of SAS
Table 1 Experimental design for Experiment 3. Semen was collected once per week and used to inseminate in vitro–matured oocytes at 6, 24, or 72 h after semen collection following storage at 5 8C*. Monday
Tuesday
Wednesday
IVM Rep 1
Semen collection IVF Rep 1 (6 h) IVM Rep 2
IVF Rep 2 (24 h)
IVM, oocyte collection and in vitro maturation; IVF, oocyte in vitro fertilization; Rep, replicate. * Ovaries were only available from abattoirs on Monday, Tuesday, and Thursday each week.
Thursday
Friday
IVM Rep 3
IVF Rep 3 (72 h)
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following square-root transformation of the sperm count. The model had fixed effects for diluent, storage time, position along the capillary tube, and all two-way interactions, and replicate by time as a random term. In vitro fertility data for proportion of oocytes that cleaved, proportion that reached the blastocyst stage on Days 6, 7, 8, and 9, and the proportion of blastocysts that hatched were transformed (arcsine) prior to analysis using Proc MIXED of SAS and a model with fixed effects for treatment and storage time and their interaction, and a random term for oocyte batch was fitted. Differences among means were evaluated using the Tukey adjustment for multiple comparisons. In vivo fertility was analyzed using Proc GENMOD of SAS with a logit link function used to fit a model that included effects for treatment, ewe breed, and replicate. Because of the small number of Cambridge and Belclare Texel ewes and the fact that the Belclare and Cambridge ewes included carriers of major genes for ovulation rate [21], these classes were combined for analysis to form a group with a high natural ovulation rate. The initial model included two-way interaction terms, but these were dropped from the final model as none approached statistical significance.
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Table 2 Mean estimates of sperm motility and viability after storage of fresh ram sperm at 5 or 15 8C for 72 h postcollection (Experiment 1). Diluent
Temperature, 8C
Motility*
Viability, %
Skim milk AndroMed OviPro INRA 96 s.e. Skim milk AndroMed OviPro INRA 96 s.e.
5 5 5 5
2.3 3.2 3 2.5 0.21 0 1.16 0.16 0.16 0.23
68.0 62.7 58.2 55.7 2.91 31.8 35.7 26.8 31.3 3.47
15 15 15 15
s.e. = standard error. * Motility was subjectively assessed using a phase-contrast microscope at 200 magnification and a 6-point scale (0 = nonmotile to 5 = rapid motility).
analyses of the data for each storage temperature. When sperm were stored at 15 8C, there was a significant diluent by time interaction for viability. This effect was not significant when storage was at 5 8C. The pattern of results for motility was similar; there was a significant time by diluent interaction when sperm were stored at 15 8C, but this effect was not significant at 5 8C.
3. Results
3.2. Experiment 2
3.1. Experiment 1
There was a significant diluents by time interaction. The number of spermatozoa penetrating mucus differed among diluents at 6 and 24 h postcollection, but no differences were evident at 48 or 72 h (Fig. 2). Among the diluents tested, the number of spermatozoa stored in INRA 96 penetrating the mucus remained relatively constant across all time points.
The residual correlation between the proportion of live sperm (viability) in duplicate aliquots was 0.28; the mean value was used in the final analysis. Storage temperature had a highly significant effect on both motility and viability; at 5 8C, viability remained relatively stable between 24 and 72 h postcollection, whereas at 15 8C, viability declined in a linear fashion as time of storage increased (Fig. 1). The estimates for motility score and viability percentage at 72 h postcollection are presented in Table 2 based on separate
Fig. 1. Proportion of live sperm up to 72 h after semen collection following semen storage at 5 8C (—) or 15 8C (– –) in skim milk (^), OviPro (&), AndroMed (~), or INRA 96 (*) (Experiment 1).
3.3. Experiment 3 Results of the preliminary experiment are shown in Table 3. The proportion of oocytes undergoing cleavage
Fig. 2. Least square means for the effect of storage diluent on the number of fresh ram spermatozoa penetrating artificial mucus at 6, 24, 48, and 72 h postcollection (Experiment 2).
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Table 3 Cleavage and development to the blastocyst stage after IVF with fresh ram sperm stored in INRA 96 for 6, 24, or 72 h at 15 8C (Experiment 3). Time, h
Number of oocytes
Cleavage, % (95% confidence interval)
Blastocyst rate, % (95% confidence interval)
6 24 72
96 107 124
74.5 (61.2–85.8) 83.5 (71.7–92.6) 2.6 (0.0–9.0)
20.9 (9.8–34.7) 27.2 (14.7–41.9) 1.0 (0.3–6.3)
Table 4 Cleavage and blastocyst development after IVF with fresh ram sperm stored at 5 8C for 6, 24, or 72 h postcollection in one of three diluents. Time, h
Diluent
Number of oocytes
Cleavage rate, % (95% confidence interval)
Blastocyst rate, % (95% confidence interval)
6
Skim milk AndroMed INRA 96 Skim milk AndroMed INRA 96 Skim milk AndroMed INRA 96
195 181 178 321 318 332 291 268 282
32.7 42.7 37.1 63.5 71.6 63.3 59.1 60.6 62.7
10.5 (3.1–21.8) 9.6 (2.5–20.5) 9.2 (2.3–20.0) 25.2 (13.2–39.7) 34.7 (20.9–50.0) 19.7 (9.0–33.3) 22.1 (11.6–34.8) 15.0 (6.4–26.4) 21.7 (11.3–34.4)
24
72
(10.8–59.7) (18.0–69.6) (13.8–64.2) (38.8–85.0) (47.4–90.6) (38.6–84.8) (34.4–81.5) (35.8–82.7) (37.9–84.3)
after IVF and the proportion developing to the blastocyst stage are presented in Table 4. After storage at 5 8C, cleavage rate was unaffected by the duration of storage (P = 0.2) or diluent (P = 0.3), and there was no evidence for an interaction (P = 0.8). In the case of blastocyst rate, there was some evidence for an effect of diluent (P = 0.08) and a significant time by diluent interaction reflecting the numerical superiority of AndroMed at the 24-h time point, whereas this diluent was poorest at 72 h, with little difference among the diluents at the 6-h time point. 3.4. Experiment 4 Sperm motility was lower at 48 and 72 h compared with that for fresh (0 h) and at 24 h, consistent with the
Fig. 3. Pregnancy rate in ewes after cervical AI using fresh ram semen stored for 0, 24, 48, or 72 h (vertical bars represent the 95% confidence interval for the mean) (Experiment 4). The estimated means (95% confidence interval) for pregnancy rate (%) were 60.2 (46.3 to 72.7), 52.2 (39.9 to 64.1), 30.4 (20.1 to 42.1), and 18.3 (10.9 to 29.4) for 0, 24, 48, and 72 h, respectively.
results of Experiment 1 for semen stored in INRA 96 at 5 8C (data not shown). Pregnancy rate, adjusted for effects of breed and replicate, are presented in Fig. 3. Pregnancy rate decreased up to 72 h after semen collection (P < 0.001). The decline was essentially linear (P < 0.001) as the quadratic term did not approach significance. The effects of ewe breed and replicate were also significant. The estimated breed means (95% confidence interval) for pregnancy rate (%) were 25.5 (10.0 to 51.1), 34.1 (17.0 to 56.6), 64.2 (54.2 to 73.1), 50.0 (40.4 to 58.9), and 35.6 (23.0 to 50.7) for Texel, Suffolk, Scottish Blackface, Belclare Scottish Blackface, and High ovulation rate groups, respectively. There was no evidence for any breed by treatment or replicate by treatment interactions. A total of 76 ewes yielded litter size data, and ovulation rate information was available for 169 ewes. There was no significant association between treatment and ovulation rate assessed postmortem (Table 5). Also, there was no evidence that treatment affected the litter size of ewes that conceived to AI. Information on score for mucus secretion (0 = none to 3 = copious) evident at insemination and the depth to which it was possible to insert the insemination pipette into the cervical canal (penetration score, 0 = no penetration to 3 = deep penetration) were recorded at the time of AI. The distribution of these scores is shown in Fig. 4. The analyses of the scores is summarized in Table 6. There was no evidence for any effect of breed or treatment on either mucus or penetration scores. The repeatability estimates for mucus and penetration scores
L. O’Hara et al. / Theriogenology 73 (2010) 541–549 Table 5 Ovulation rate and litter size for ewes in AI study (Experiment 4). Factor
Ovulation rate*
Litter size
Treatment (time, h) 0 24 48 72 F-test
2.37 0.179 2.21 0.158 2.49 0.162 2.07 0.176 NS
1.79 0.204 1.68 0.206 1.77 0.251 1.45 0.338 NS
Ewe breed Texel Suffolk Scottish Blackface Belclare Scottish Blackface High ovulation rate group F-test
1.79 0.294 2.07 0.263 1.72 0.112 2.28 0.109 3.44 0.176 P < 0.001
1.48 0.362 1.07 0.415 1.56 0.132 1.68 0.142 2.21 0.235 P = 0.109
NS, not significant. * Adjusted for pregnancy status.
were 0.23 and 0.22, respectively (P < 0.01). There was no evidence (P = 0.6) for any association between penetration score and pregnancy rate, but a positive association between pregnancy rate and mucus score approached formal significance (P = 0.08; Proc GENMOD with mucus score as a covariate). 4. Discussion This study involved the evaluation of the effect of storage of fresh ram semen for up to 72 h postcollection in terms of sperm motility and viability as well as more functional measures including the ability of sperm to penetrate artificial mucus, to fertilize oocytes in vitro, and ultimately to establish pregnancy in ewes after cervical insemination. The main findings are (1) fresh storage of ram semen at 5 8C is superior to storage at 15 8C, (2) a high proportion of sperm retain their ability to penetrate artificial mucus for up to 72 h, (3) storage at 5 8C for 72 h postcollection does not diminish the ability of sperm to fertilize oocytes in vitro, and (4)
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increased duration of storage is associated with a linear decrease in pregnancy rate in ewes after AI. The liquid storage of ram semen has been extensively reviewed in recent years [13,22]. Irrespective of the diluent used, dilution rate, temperature, or conditions of storage, the quality of spermatozoa deteriorates as the duration of storage increases. The main changes that occur during storage include a reduction in motility, morphologic integrity, and fertility of spermatozoa. It is hypothesized that these changes are accompanied by a decline in the survival of spermatozoa in the female reproductive tract and may be associated with an increase in embryonic loss. Although several groups have assessed the effect of storage on motility and viability [2,7], relatively few have translated the in vitro observations to field fertility [23]. This is important because there are a plethora of studies in the literature examining the ability to predict field fertility from laboratory tests in both sheep [19,24] and cattle [25,26]. Most of these results are equivocal. Traditional in vitro evaluation of semen quality involves the subjective assessment of motility and the proportion of sperm with normal morphology. Other, more elaborate tests include measures of viability, acrosomal integrity, objective computer-based assessment of motility, and IVF. Whereas some success has been achieved using these measures, few, if any, single in vitro tests show a reliable and repeatable correlation with field fertility. In Experiment 1, storage temperature had a significant effect on sperm motility and viability up to 72 h postcollection, irrespective of diluent; storage at 15 8C was detrimental to both parameters, whereas at 5 8C both motility and viability remained relatively constant. Although motility is an important measure of sperm functionality, it is difficult to relate this parameter to field fertility. Sperm can retain their motility in vitro for a relatively long period of time. For example, LopezSaez et al. [2] examined the effect of three diluents
Table 6 Ewe breed effects on scores from mucus and penetration at AI (Experiment 4).
Fig. 4. Frequency distribution for mucus and depth of penetration scores at the time of cervical insemination (Experiment 4).
Ewe breed
Penetration score
Mucus score
Suffolk Texel Scottish Blackface Belclare Scottish Blackface High ovulation rate group F-test
1.8 0.17 1.4 0.20 1.4 0.08 1.5 0.07 1.4 0.12 NS
2.2 0.09 1.9 0.10 2.0 0.04 2.1 0.04 2.0 0.06 NS
NS, not significant.
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(skim milk and two Tris-based extenders) on ram semen quality in terms of motility and membrane integrity after storage at 5 8C for up to 16 d. Whereas the percentage motile sperm decreased with time, a significant proportion (>40%) was still motile after 16 d storage in the two Tris-based diluents. Similarly, a proportion of sperm were capable of fertilizing oocytes in vitro and in vivo after storage at 5 8C for 7 d and even 14 d [27,28]. The current findings that storage at 5 8C was associated with only minor changes in motility and viability up to 72 h compared with the major decline when storage was at 15 8C are consistent with those of Paulenz et al. [7] who reported similar results for motility, along with acrosome integrity and capacitation status after 0 to 30 h storage of liquid ram semen at 5 8C compared with storage at 20 8C. The ability of sperm to move through the mucus-rich environment of the female reproductive tract, particularly the cervix, was assayed by assessing sperm migration in artificial mucus [20]. In comparison with cervical mucus, artificial mucus provides a standard medium with no biological variability allowing standardized assessment of migration. Gillan et al. [20] showed that spermatozoa from bulls with high fertility migrate through such mucus in larger proportions and that the vanguard spermatozoon migrated a greater distance than spermatozoa from low-fertility bulls. The number of spermatozoa penetrating mucus in the current study differed slightly among diluents at 6 and 24 h postcollection but was relatively constant at 48 and 72 h. Among the diluents tested, the number of spermatozoa stored in INRA 96 penetrating the mucus remained relatively constant across all time points. It has been stated that one of the constraining factors in the use of fresh stored ram semen at 15 8C is that the sperm fertilizing ability declines with time (6 to 12 h) [2]. Our results would contradict this notion somewhat; whereas storage for 72 h at 15 8C resulted in a dramatic reduction in the proportion of oocytes undergoing cleavage and, as a consequence, developing to the blastocyst stage, the fertilizing ability of the sperm was unaffected up to 24 h postcollection. Furthermore, irrespective of diluent used, neither cleavage rate nor blastocyst development decreased up to 72 h postcollection when storage was at 5 8C (Table 4). Maxwell and Stojanov [27] reported that a proportion of sperm were capable of fertilizing oocytes in vitro after storage at 5 8C for 7 d and even 14 days. The increase in oocyte cleavage after IVF and blastocyst development between 6 and 24 h of storage in the current study is probably a reflection of the suboptimal or incomplete capacitation status of the sperm 6 h after collection.
Successful fertilization in vivo depends not only on the performance of the sperm but also on the transport of sperm and site of deposition in the female reproductive tract. After confirming that fresh ram semen stored at 5 8C, irrespective of diluent, was capable of fertilizing oocytes up to 72 h postcollection, the fertilizing ability of such semen in vivo was examined. In studies reviewed by Maxwell and Salamon [22], fertility declined rapidly when semen stored for more than 24 h was used for cervical insemination (by 10% to 35% per day of storage); lambing rates for semen stored for 24, 48, and 72 h were 45% to 50%, 25% to 30%, and 15% to 20%, respectively. It is known that the cervix represents a formidable obstacle that sperm must traverse after natural mating or cervical insemination. With compromised sperm (i.e., frozen-thawed [14] or chilled-stored [28]), pregnancy rates are higher after intrauterine insemination compared with those after cervical insemination suggesting that issues related to sperm transport in the female reproductive tract play a significant role in the decline in fertility seen with such spermatozoa. Salamon et al. [28] showed that the fertilizing capacity of ram spermatozoa is retained for up to 10 d of storage at 5 8C, but lambing rates after cervical insemination declined substantially when the semen was stored for more than 3 d. In the study of Maxwell and Stojanov [27], the in vivo fertilization and lambing rates obtained after 7 d storage of semen in the presence of antioxidants were not higher than those reported by Salamon et al. [28] after 8 d, but the storage time for which the spermatozoa retained their fertilizing capacity (14 d) was beyond previous limits. Furthermore, pregnancies were obtained after intrauterine insemination with semen chilled-stored for 14 d. In the current study, semen stored in INRA 96 at 5 8C gave acceptable pregnancy rates after insemination up to 24 h postcollection (63% and 54% for 0 and 24 h, respectively). Pregnancy rates decreased after 24 h, with rates of 30% and 20% for 48 and 72 h stored semen, respectively. These results are similar to those reviewed by Maxwell and Salamon [22]. Ewe breed had a significant effect on pregnancy rate. This effect was unexpected, as generally ewe breed has not been reported as a source of variation in conception rate after AI with fresh semen. It has, however, been a factor in the success of conception rate after the use of frozen-thawed semen in AI [4,5]. In conclusion, the results of this study indicate that ram semen cooled and stored at 15 8C has a shorter life span than that cooled to 5 8C. Furthermore, the commercially available diluents tested in this study did not differ significantly in terms of fresh ram semen
L. O’Hara et al. / Theriogenology 73 (2010) 541–549
performance in vitro when stored at 5 8C. Sperm storage in INRA 96 at 5 8C gave acceptable pregnancy rates up to 24 h after semen collection, and given that it can be used directly off the shelf, it could offer an alternative to the standard skim milk currently used. Where possible, cervical insemination should be performed within 24 h of semen collection to obtain acceptable pregnancy rates. In situations where this is not possible, semen stored at 5 8C can still be used for cervical insemination up to 3 d postcollection, although pregnancy rates will be decreased. Acknowledgments We acknowledge the technical staff at Teagasc Research Centre, Athenry, Ireland, for management of rams and collection of semen. We would also like to acknowledge Mary Wade at Lyons Research Farm, UCD, Newcastle, Ireland, for media preparation. This work was funded by Teagasc under the Walsh Fellowship Scheme. References [1] Fair S, Hanrahan JP, Donovan A, Duffy P, O’Meara CM, Lonergan P, Evans AC. Hormonal relationships during the periovulatory period among ewe breeds known to differ in fertility after cervical artificial insemination with frozen thawed semen. Anim Reprod Sci 2007;97:284–94. [2] Lopez-Saez A, Ortiz N, Gallego L, Garde JJ. Liquid storage (5 degrees C) of ram semen in different diluents. Arch Androl 2000;44:155–64. [3] Vishwanath R, Shannon P. Storage of bovine semen in liquid and frozen state. Anim Reprod Sci 2000;62:23–53. [4] Donovan A, Hanrahan JP, Lally T, Boland MP, Byrne GP, Duffy P, et al. AI for Sheep Using Frozen-Thawed Semen. End of Project Report: Sheep Series No. 11. Teagasc, Agriculture and Food Development Authority, 2001. [5] Donovan A, Hanrahan JP, Kummen E, Duffy P, Boland MP. Fertility in the ewe following cervical insemination with fresh or frozen-thawed semen at a natural or synchronised oestrus. Anim Reprod Sci 2004;84:359–68. [6] Eppleston J, Maxwell WMC. Recent attempts to improve the fertility of frozen ram semen inseminated into the cervix. Wool Technology and Sheep Breeding 1993;41:291–302. [7] Paulenz H, Soderquist L, Perez-Pe R, Berg KA. Effect of different extenders and storage temperatures on sperm viability of liquid ram semen. Theriogenology 2002;57:823–36. [8] Bailey JL, Bilodeau JF, Cormier N. Semen cryopreservation in domestic animals: a damaging and capacitating phenomenon. J Androl 2000;21:1–7. [9] Byrne GP, Lonergan P, Wade M, Duffy P, Donovan A, Hanrahan JP, Boland MP. Effect of freezing rate of ram spermatozoa on subsequent fertility in vivo and in vitro. Anim Reprod Sci 2000;62:265–75. [10] Maxwell WMC, Watson PF. Recent progress in the preservation of ram semen. Anim Reprod Sci 1996;42:55–65.
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