Influence of extender, temperature, and addition of glycerol on post-thaw sperm parameters in ram semen

Theriogenology 59 (2003) 1241±1255

In¯uence of extender, temperature, and addition of glycerol on post-thaw sperm parameters in ram semen Jorge Gila,b, Nils Lundeheimb,c, Lennart SoÈderquista,b, Heriberto RodrõÂguez-MartõÂneza,b,* a

Department of Obstetrics and Gynecology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences (SLU), UllsvaÈgen 14C, Box 7039, Uppsala SE-750 07, Sweden b Center for Reproductive Biology in Uppsala (CRU), Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden c Department of Animal Breeding and Genetics, Faculty of Agriculture, Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden Received 13 September 2001; accepted 10 June 2002

Abstract Using a two-step extension methodology, two experiments were conducted using a split-sample design to compare the effect on post-thaw ram sperm parameters of a milk-based extender (Experiment 1) containing four different egg yolk concentrations (5% [M5], 10% [M10], 15% [M15], and 20% [M20]), and a commercially available extender (Bioexcell1; IMV, L'Aigle, France) free from additives of animal origin, containing two different ®nal glycerol concentrations (3.2% [B] and 6.4% [BB]) (Experiment 2). In both experiments, glycerol was added either at 5 8C or at 15 8C together with the second fraction of each extender. The sperm characteristics assessed were motility (measured subjectively [SM] and by means of cell motion analysis (CASA)), membrane integrity (SYBR-14/PI), and capacitation status (chlortetracycline (CTC)/EthD-1). Results of Experiment 1 showed no signi®cant positive effect of increasing the concentration of egg yolk above 10% on post-thaw motility, membrane integrity, or induction of sperm capacitation-like changes. In Experiment 2, Bioexcell1 (BB) yielded similar post-thaw results as did the milk extender (control). In both experiments, post-thaw sperm parameters were better preserved when glycerol was added at 5 8C, although the results were not always statistically signi®cant for all variables studied. In conclusion, when using milk-based extenders for freezing ram semen, low (5±10%) concentrations of egg yolk and the addition of glycerol at 5 8C are recommended. Furthermore, the results indicate that when freezing ram semen, Bioexcell1 containing 6.4% * Corresponding author. Tel.: ‡46-186-72172; fax: ‡46-186-73545. E-mail address: [email protected] (H. RodrõÂguez-MartõÂnez).

0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 1 1 7 7 - 9

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glycerol may be used as an alternative extender to the conventional milk extender containing 5% egg yolk. # 2002 Elsevier Science Inc. All rights reserved. Keywords: Capacitation; Extender; Post-thaw; Ram; Semen; Viability

1. Introduction The sheep industry is interested in improving long-term semen preservation, particularly for use in cervical arti®cial insemination (AI), an approach that is more practical than intrauterine deposition of semen [1±3]. However, the relatively low success rate of cervical AI with frozen semen in sheep has limited a wider application of this technique, calling for an improvement of the insemination technique itself, and of the survival rate of the frozen± thawed semen. Suitable extenders to preserve an adequate number of spermatozoa with all the attributes needed to overcome the cervical barrier and to achieve fertilization postthawing, are needed to achieve these goals [4±7]. In Norway and Sweden, the routine protocol for freezing ram semen consists of a twostep extension method using a milk-based extender. First, the semen is diluted with an extender without glycerol (fraction 1 (f/1)), and cooled to 5 8C, then further diluted 1:1 with an extender (fraction 2 (f/2)) containing a glycerol concentration of 14%, resulting in a ®nal concentration of 7%. It is equilibrated for 2 h, centrifuged to re-concentrate the sperm concentration to 200  106 spermatozoa/AI dose, packed in 0.25 ml mini-straws, and ®nally frozen in LN2 [8]. We have previously studied in vitro as well as in vivo the effect of an adjustment of the extension rate of the semen as an alternative technique that would exclude the centrifugation step, thus simplifying the processing routines and avoiding the stress of centrifugation on spermatozoa [9,10]. Our results indicated that the quality of the AI dose was maintained, and that despite the prefreezing semen/extender ratio being below that known as optimal for spermatozoa [11±13], fertility was maintained at acceptable levels. However, possible ways of reducing other eventual negative effects of a low semen/extender ratio would be to increase the amount of known protective ingredients in the extender, such as egg yolk, or the addition of a cryoprotectant at an earlier step during cooling, for instance before the temperature reaches 5 8C. Several studies [14±16] have shown that cold shock starts soon after the semen is cooled to room temperature, although it is more critical for ram semen at temperatures below 15 8C [17]. Glycerol, despite its value as cryoprotectant, is metabolically toxic to spermatozoa, and noxious to membrane integrity, depending on the concentration and the temperature at which it is added, thus calling for a compromise if optimal results are to be achieved [18±21]. Using animal-derived additives such as milk or egg yolk in a semen extender implies sanitary risks, not only through the inclusion of speci®c microbiological agents, but also by contaminants that may compromise the quality of the product [22,23]. This topic, frequently discussed in the cattle AI industry, is of utmost importance in sheep AI as well. An alternative to egg yolk in extenders for ram semen may be soybean lecithin, present in a commercial extender developed for bull semen (Bioexcell1; IMV, L'Aigle, France).

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Therefore, the objective of the present work was to compare the effect of a milk-based extender (Experiment 1), containing four different egg yolk concentrations (5, 10, 15, and 20%, respectively), with that of the commercial Bioexcell1 extender, containing two different ®nal glycerol concentrations (3.2 and 6.4%), on post-thaw ram sperm characteristics. In both experiments, the effect of adding glycerol at either 5 or 15 8C together with the second fraction of each extender was also studied. 2. Materials and methods 2.1. Animals and semen collection 2.1.1. Experiment 1 In this experiment, semen from ®ve crossbred rams, 10±12 months of age, was used. The rams were housed at the Department of Obstetrics and Gynecology, at the Swedish University of Agricultural Sciences (SLU), Uppsala, for 2 months during the breeding season (October to November). Semen was collected daily using an arti®cial vagina, beginning 1 month before the start of the experiment. Two consecutive ejaculates were collected within 10 min, and later pooled and treated as a single ejaculate. The ejaculates were within normal ranges regarding volume (0.75±2 ml), concentration ( 2:5  109 spermatozoa/ml), motility (70%), and morphology (10% total sperm abnormalities). 2.1.2. Experiment 2 Semen from ®ve rams (®ve different breeds), 20±24 months of age, was used in this experiment. The rams were housed at the Swedish Sheep Farmers Associations ram AI station (KungsaÈngen, Uppsala) and involved in a daily semen collection routine during the northern hemisphere breeding season (October to November). Only the second ejaculates, collected over 5 consecutive days in November, were used. As in Experiment 1, all ejaculates used were within the ranges of normality, as stated earlier. 2.2. Extenders All extenders were prepared as follows, and stored at the experiments.

18 8C until thawed and used in

2.2.1. Experiment 1 The effect of four milk-based extenders (M1, M2, M3, and M4) was studied. Because of the two-step extension methodology used, each of the four extenders comprised two fractions, the ®rst without glycerol and the second with 14% (v/v) glycerol, thus resulting in a ®nal concentration of 7% (v/v) glycerol. Extenders were prepared from nonfatty milk powder (11%, w/v) and distilled water. The milk was heated to 95 8C for 10 min on a stirring plate equipped with a heater and then cooled to room temperature for further processing. Fraction 1 was prepared by adding enough egg yolk to the milk-based extender to reach concentrations of 5% (M5), 10% (M10), 15% (M15), and 20% (M20) (v/v). After homogenization of the milk±egg yolk extender, all f/1 preparations were

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centrifuged for 20 min at 3310  g at 5 8C to clarify the extender by removing large particles and overlaying lipids. The clari®ed supernatant was supplemented with antibiotics (penicillin 0.03 g/100 ml and streptomycin 0.04 g/100 ml). All f/2 were prepared by adding 224 mM of fructose, egg yolk (5, 10, 15, 20%, v/v), and glycerol (14%, v/v) to the milk base prepared, and supplemented with antibiotics, as described earlier. 2.2.2. Experiment 2 Three extenders were used. The ®rst extender was a milk-based extender (M), prepared as M5 in Experiment 1, and used as the control. The second extender (B) was the Bioexcell1 extender (IMV, L'Aigle, France), which is commercially available for bull semen. Due to the need for a two-step extension procedure, special preparations were manufactured (IMV, L'Aigle, France), with f/1 without glycerol and f/2 with 6.4% (v/v) glycerol. The third extender (BB) was the same as extender B, except that the f/2 contained 12.8% (v/v) glycerol. 2.3. Semen processing After collection, the ejaculates were placed in a portable waterbath at 33 8C. An aliquot from each ejaculate was taken to measure the sperm concentration in a photometer (SpermaCue; MinituÈb, Tiefenbach, Germany). Each ejaculate from each ram was initially split into as many fractions as needed for the experiments (Experiment 1 ˆ 4, Experiment 2 ˆ 3) (Fig. 1), and then extended 1 ‡ 1 with f/1 of extenders M5, M10, M15, and M20, respectively (Experiment 1), and with f/1 of extenders M, B, and BB, respectively (Experiment 2). After photometrically measuring the sperm concentrations, aliquots of the preliminary extended semen, with equal sperm numbers from each ram, were then pooled within each extender, and the pools were further extended to 1:6  109 cells/ml with f/1 of each extender. After careful homogenization of the primary pools, the extended semen was again split into two secondary pools, and further processed according to one of the following protocols (see Fig. 1). Protocol 5 8C (P5 8C): 1. 2. 3. 4. 5.

Cooling to 5 8C within 60 min in a waterbath. Dropwise extension at 5 8C with f/2 of each extender to 0:8  109 cells/ml. Equilibration at 5 8C for 2 h. Packaging in 0.25 ml mini-straws at 5 8C. Freezing. Protocol 15 8C (P15 8C):

1. 2. 3. 4. 5. 6.

Cooling to 15 8C within 30 min. Dropwise extension at 15 8C with f/2 of each extender to 0:8  109 cells/ml. Further cooling to 5 8C within 30 min in a waterbath. Further equilibration at 5 8C for 1.5 h. Packaging in 0.25 ml mini-straws at 5 8C. Freezing.

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Fig. 1. Experimental design. In Experiment 1, the effect on sperm quality after thawing was studied with a split-sample design (4  2 layout), using four extenders (M5, M10, M15, and M20) and addition of the glycerolized fraction at either 5 or 15 8C. In Experiment 2, three extenders (M, B, and BB) were evaluated using the same two protocols as in Experiment 1. The procedure shown in ®gure was repeated ®ve times in both experiments.

For practical reasons, during transport from the ram AI station to the freezing laboratory, the secondary pools were cooled to 15 8C within 30 min, either in a waterbath (Experiment 1) at the freezing laboratory (Department of Obstetrics and Gynecology, SLU) or in a portable isothermic chamber with wrapped freeze-packs (Experiment 2) to mimic the cooling rate of the waterbath. The freezing was done in a programmable freezing chamber (Digitcool 5300; IMV, L'Aigle, France). Here, the temperature was lowered from 5 to 8 8C at a rate of 3 8C/min, and from 8 to 130 8C at 25 8C/min. The straws were then transferred to LN2 for storage until evaluated. In total, ®ve freezing operations for each experiment were done. 2.4. Experimental design Two experiments were performed using a split-sample design. In Experiment 1 (4  2 layout), the effect on sperm quality post-thaw was studied using four extenders (M5, M10, M15, and M20) with the glycerolized fraction being added at 5 8C (P5 8C) or at 15 8C (P15 8C), respectively (Fig. 1). In Experiment 2 (3  2 layout), three extenders (M, B, and BB) were studied using the same two protocols for the addition of the glycerolized fraction of each extender.

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2.5. Post-thaw semen evaluation 2.5.1. Thawing of the straws The straws were thawed at 50 8C for 9 s in a waterbath. For each evaluation, three straws were thawed and pooled in a test tube. 2.5.2. Subjective motility (SM) This was assessed post-thaw using a phase contrast microscope equipped with a warm stage (38 8C) (Nikon, Tokyo, Japan). Ten microliters of pooled semen was diluted at 38 8C with 100 ml of sucrose buffer (10 mM NaCl, 222 mM sucrose, 2.5 mM KCl, 20 mM HEPES, 10 mM glucose, and 1 mg/100 ml of cold water-soluble polyvinyl alcohol (pH 7.5 with NaOH 1N; Sigma Aldrich, TyresoÈ, Sweden)). Then, 5 ml of the diluted sample was placed in a Makler Counting Chamber (Se®-Medical Instruments, Haifa, Israel), and the frequency of sperm motility assessed to the nearest 5%, after judging four different microscopic ®elds. 2.5.3. Cell motion analysis (CASA) For the same preparation of diluted semen and with the same microscope as used for SM (see earlier description), CASA was performed with a computer-assisted motility analysis system (StroÈmberg-Mika Cell Motion Analyzer; SM-CMA, MTM Medical Technologies, Montreux, Switzerland). For each evaluation, eight microscopic ®elds (sequences) were analyzed to include at least 200 spermatozoa. The proportion of total motile (TM) and linearly motile (LM) spermatozoa, straight-line velocity (VSL, mm/s), curvilinear velocity (VCL, mm/s), average-path velocity (VAP, mm/s), and spermatozoa with lateral head displacement (LHD, mm) was determined. The main software settings were 32 frames per sequence, minimum of 15 frames per object, 10 mm/s as a velocity limit for immobile objects, 25 mm/s as a velocity limit for local motile objects, and 25 mm for the maximum radius of circles. 2.5.4. Sperm membrane integrity This was assessed using staining with SYBR-14 and propidium iodide (SYBR-14/PI; Molecular Probes Inc., Eugene, OR, USA), as described by Garner and Johnson [24]. Thawed semen (50 ml) was mixed with 5 ml of SYBR-14 and 2.5 ml of PI (10 mM SYBR-14 and 120 mM PI). The sample was then gently mixed and incubated at 37 8C for 15 min. The smears were prepared by placing 4 ml of stained semen and 1 ml of glutaraldehyde solution (3% electron microscopy grade in cacodylate buffer, pH 7.4) on a warm slide just before the assessment in order to stop the motion, thus facilitating counting. Two hundred spermatozoa were counted using a microscope (Leitz Laborlux-11; Jena, Germany) equipped with a Paralens1 objective set (60, 470±490 nm excitation ®lter, 510 nm dichroic beam splitter, 520 nm barrier ®lter; Becton-Dickinson, Leiden, The Netherlands) and classi®ed as `intact' when stained green or as `membrane damaged' when stained green±red or just red. 2.5.5. Capacitation status This was assessed in viable spermatozoa with chlortetracycline (CTC), as described by Gil et al. [9,10,25]. To assess only the viable spermatozoa, 18 ml of thawed spermatozoa

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was incubated for 10 min at 37 8C with 2 ml of ethidium homodimer (23.3 mM EthD-1, Molecular Probes Inc., Eugene, OR, USA). After incubation, the mixture was ®xed with 20 ml of 3% glutaraldehyde (electron microscopy grade in cacodylate buffer, pH 7.4), and then stained with 40 ml of CTC working solution. The sample was washed by centrifugation at 700  g for 8 min with 1 ml of sucrose buffer (see earlier description). Thereafter, 1 ml of the supernatant was removed and the pellet re-suspended. The smears were prepared with 4 ml of stained sample plus 2 ml of antifade (0.1% p-pheneylendiamine in 9 ‡ 1 of glycerol and phosphate-buffered saline (PBS)), mixed on a clean microscope slide, covered with a cover glass, sealed with nail varnish, and kept in the dark at 4 8C. The evaluations were done, within 12 h of staining, using a microscope (Leitz Diaplan-20, Jena, Germany) with epi¯uorescent optics and a set of violet±blue ®lters (420±490 nm excitation, 510 nm emission) and green ®lters (530±560 nm excitation, 580 nm emission). Two hundred viable spermatozoa (unstained with EthD-1) were classi®ed into three categories: the uniform ¯uorescent head (uncapacitated: CTC-F), the ¯uorescence-free band in the post-acrosomal region (capacitated: CTC-B), and the non¯uorescent head or a thin ¯uorescent band in the equatorial segment (acrosome-reacted: CTC-AR) [9,26]. 2.6. Statistical analysis The results of each experiment were analyzed using the General Linear Model (GLM) in the Statistical Analysis System package (SAS Institute, Inc., Cary, NC, USA, 1996). The results are presented as least square means …LSM†  standard error of the means (S.E.M.). In the statistical model, the classes de®ned were the freezing operation, extender, freezing protocol, and extender  freezing protocol interaction. Due to the signi®cant differences found for the freezing operation in Experiment 1, a further mixed procedure was used including the effect of the freezing operation as a random effect. 3. Results 3.1. The effect of extenders on sperm parameters 3.1.1. Experiment 1 For motility parameters, the SM values (%) obtained were higher (although NS) for M5 and M10 than for M15 and M20 (70  2:2, 70  2:2, 68  2:2, and 67  2:2, respectively). The TM values (%) were almost equal for all extenders (83  2:3, 83  2:3, 84  2:3, and 83  2:3), as were the frequencies of LM spermatozoa assessed by CASA (M5: 65  2:8%, M10: 62  2:8%, M15: 60  2:8%, and M20: 59  2:8%). The velocity patterns (VSL, VCL, VAP, and LHD) assessed did not differ signi®cantly between extenders. The percentages of spermatozoa with intact membranes (SYBR-14/PI) were lower in M5 and M20 than in M10 and M15 (M5: 64  2:1, M10: 69  2:1, M15: 66  2:1, M20: 63  2:1). The overall effect of the extenders on membrane intactness (P < 0:01) was affected by the extender used, and the comparison between extenders showed statistically signi®cant differences between M5 and M10 and between M10 and M20 (P < 0:05 to P < 0:01).

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The percentage of uncapacitated spermatozoa assessed by CTC (CTC-F) was signi®cantly affected (P < 0:01) by the extender used. The highest value was seen in M5 (51  2:4%) compared with the other extenders (M10: 41  2:4%, M15: 39  2:4%, and M20: 41  2:4%). Conversely, the population of capacitated spermatozoa (CTC-B) was lower in M5 extender (44  2:6%; P < 0:01) than in all the others (M10: 51  2:6%, M15: 53  2:6%, and M20: 52  2:6%). The percentage of acrosome-reacted spermatozoa (CTC-AR) did not differ between extenders. 3.1.2. Experiment 2 Overall, the motility (SM, TM, and LM) was signi®cantly (P < 0:001) in¯uenced by extenders. The pairwise test of SM showed signi®cant differences between extenders B (26  2:9%) and M and BB, but not between M and BB (58  2:9 and 53  2:9%, respectively). The percentage of TM spermatozoa was signi®cantly (P < 0:001) lower in B (28  4:2%) than in M (67  4:2%) and BB (63  4:2%). The percentages of LM spermatozoa were signi®cantly (P < 0:001) lower for extender B (21  3:3%) than for BB (45  3:3%) and M (44  3:3%). The VSL values differed signi®cantly (P < 0:001) between extenders BB and B as well as between BB and M, but not between B and M, with a higher value for B (115  3:8) than for M and BB (114  3:8 and 102  3:8, respectively). The VAP (mm/s) for extender M was higher (126  4:1) than for B (123  4:1) and BB (111  4:1), and the difference was signi®cant (P < 0:01) between BB and the other two extenders. The VCL (mm/s) was higher for spermatozoa frozen in M (159  4:1) than for those frozen in B (141  4:1) and BB (135  4:1), and the difference was signi®cant (P < 0:01) between M and the other two extenders. Overall, the percentage of spermatozoa with intact membranes was in¯uenced by extenders (P < 0:001). The proportion was higher in samples frozen in extender M (57  3:1%) than in those frozen in extenders BB (46  3:1%) and B (33  3:1%). The pairwise comparison showed signi®cant differences (P < 0:001) between the three extenders. The proportion of CTC-F spermatozoa (uncapacitated) were also affected by extenders (P < 0:001), where extender M (44  2:4%) showed signi®cantly higher (P < 0:001) values than did BB (30  2:4%) and B (30  2:4%). No signi®cant difference was found between B and BB. Conversely, the proportion of CTC-B (i.e. capacitated spermatozoa) was lower (P < 0:001) in extender M (48  1:8%) than in BB (50  1:8%) and in B (58  1:8%). No signi®cant difference was found between M and BB. The proportion of CTC-AR spermatozoa was lower in extender M (8  1:4) than in B (11  1:4) and in BB (19  1:4), and signi®cant differences were found between all extenders (P < 0:05 to P < 0:001). 3.2. The effect of the freezing protocol on sperm parameters 3.2.1. Experiment 1 Addition of the f/2 (glycerolized fraction) at 15 8C (69  2:1%) did not affect the SM compared to addition at 5 8C (69  2:1%). The proportion of TM spermatozoa assessed by CASA did not differ signi®cantly between P5 8C and P15 8C (84  1:8% versus 82  1:8%), nor did the proportion of LM spermatozoa (60  2:4% versus 62  2:4%). No signi®cant difference in velocity patterns was found between protocols.

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The percentage of spermatozoa having intact membranes was signi®cantly higher (P < 0:01) in the samples processed using the P5 8C protocol than in those processed using P15 8C (68  1:8% versus 63  1:8%). Regarding the capacitation status, the CTC-F value was slightly higher (NS) in P5 8C than in P15 8C (44  1:9% versus 42  1:9%). The CTC-B values were equal in both protocols (50  2:3%) and the CTC-AR spermatozoa values were signi®cantly lower (P < 0:01) in P5 8C than in P15 8C (6  0:9% versus 8  0:9%). 3.2.2. Experiment 2 The addition of f/2 (with glycerol) at 5 8C yielded higher (P < 0:05) percentages of SM compared to addition at 15 8C (48  2:6% versus 42  2:6%). Furthermore, the percentage of TM spermatozoa was higher (NS) in P5 8C than in P15 8C (56  3:6% versus 51  3:6%), and the percentage of LM spermatozoa was higher (although NS) in P5 8C than in P15 8C (39  2:9% versus 35  2:9%). The velocity patterns VSL, VAP, and VCL did not differ between the two protocols for addition of glycerol. The percentage of membrane-intact spermatozoa was higher (NS) for P5 8C than for P15 8C (47  2:9% versus 44  2:9%). Finally, the percentage of CTC-F spermatozoa was slightly higher (NS) for P5 8C (36  2:2%) than for P15 8C (35  2:2%), the values for CTC-B spermatozoa were equal (52  1:6%), and the proportion of CTC-AR spermatozoa was higher (NS) in P15 8C than in P5 8C (13  1:2% versus 12  1:2%). 3.3. The effect of the interaction between freezing protocol and extender on the sperm parameters 3.3.1. Experiment 1 The extender  protocol interaction did not in¯uence either the motility (SM, LM, and TM; Table 1) or the sperm velocity parameters assessed by CASA (VSL, VAP, and VCL; Table 2). Table 1 Motility parameters in frozen±thawed ram spermatozoa (max/min (LSM)) for two freezing protocols (P5 8C and P15 8Ca) and four milk extenders (M5, M10, M15, and M20b) Extender

Protocol

Subjective motile (S.E.M. 2.5)

Total motile (CASA; S.E.M. 3.1)

Linear motile (CASA; S.E.M. 3.6)

M5

P5 8C P15 8C

74/68 (71) 75/65 (70)

89/76 (84) 92/77 (82)

69/46 (63) 78/59 (66)

M10

P5 8C P15 8C

76/65 (71) 76/66 (70)

90/78 (85) 88/70 (81)

67/52 (61) 72/56 (62)

M15

P5 8C P15 8C

78/61 (69) 74/58 (68)

94/78 (84) 89/75 (84)

75/50 (60) 68/52 (61)

M20

P5 8C P15 8C

74/58 (66) 74/58 (69)

89/69 (83) 91/67 (82)

65/49 (57) 74/48 (61)

a b

With the addition of the glycerolized fraction at 5 and 15 8C, respectively. Containing 5, 10, 15, and 20% egg yolk, respectively.

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Table 2 Velocity (mm/s) in frozen±thawed ram spermatozoa assessed by CASA (max/min (LSM)) for two freezing protocols (P5 8C and P15 8Ca) and four milk extenders (M5, M10, M15, and M20b) Extender

Protocol

Straight-line (VSL; S.E.M. 4.1)

Average-path (VAP; S.E.M. 3.9)

Circular-line (VCL; S.E.M. 3.8)

M5

P5 8C P15 8C

150/120 (138) 158/126 (142)

161/136 (151) 170/134 (151)

193/174 (184) 201/163 (180)

M10

P5 8C P15 8C

142/120 (133) 151/123 (138)

153/131 (144) 163/133 (148)

186/167 (179) 200/167 (181)

M15

P5 8C P15 8C

148/124 (133) 147/124 (138)

155/139 (145) 158/140 (150)

190/174 (179) 191/176 (184)

M20

P5 8C P15 8C

140/112 (129) 152/116 (135)

155/125 (142) 161/129 (146)

192/158 (177) 195/160 (178)

a b

With the addition of the glycerolized fraction at 5 and 15 8C, respectively. Containing 5, 10, 15, and 20% egg yolk, respectively.

The overall extender  protocol interaction for the extenders signi®cantly in¯uenced the membrane integrity (P < 0:01), and the pairwise comparison within extender showed higher proportions of spermatozoa with intact membranes for all extenders processed using the P5 8C protocol than for P15 8C, but the difference was only signi®cant for M15 (70  2:6% versus 63  2:6%; P < 0:05). Regarding the capacitation status, no difference in the CTC-F or in the CTC-B was found. There was only a tendency for a difference (P ˆ 0:06) between protocols within M15 for CTC-B (P5 8C: 57  3:2% versus P15 8C: 50  3:2%). The overall CTC-AR was higher (P < 0:01) with P15 8C than with P5 8C (6  1:0% versus 8  1:0%), mainly due to the difference found in extender M10 when Table 3 Percentages (max/min (LSM)) of frozen±thawed ram spermatozoa showing sperm membrane integrity (SYBR14/PI) and phases of capacitation (CTC) for two freezing protocols (P5 8C and P15 8Ca) and four milk extenders (M5, M10, M15, and M20b) Extender

Protocol

Membrane integrity (S.E.M. 2.6)

Uncapacitated (S.E.M. 3.1)

Capacitated (S.E.M. 3.2)

M5

P5 8C P15 8C

69/61 (65) 71/54 (63)

60/47 (53) 55/49 (48)

50/35 (42) 56/40 (47)

M10

P5 8C P15 8C

75/65 (71) 72/61 (67)

48/39 (44) 52/26 (38)

58/40 (51) 61/38 (51)

12/3 (5) 13/7 (10)**

M15

P5 8C P15 8C

82/61 (70)* 71/57 (63)

48/30 (39) 53/32 (40)

68/49 (57) 62/37 (50)

9/2 (4) 17/4 (10)**

M20

P5 8C P15 8C

70/61 (65) 70/49 (61)

44/37 (41) 48/30 (42)

58/45 (52) 61/48 (52)

12/4 (7) 9/3 (6)

a

With the addition of the glycerolized fraction at 5 and 15 8C, respectively. Containing 5, 10, 15, and 20% egg yolk, respectively. * Denotes signi®cant difference (P < 0:05) within extender between protocols. ** Denotes signi®cant difference (P < 0:01) within extender between protocols. b

Acrosome-reacted (S.E.M. 1.4) 8/3 (5) 7/3 (5)

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Table 4 Motility parameters in frozen±thawed ram spermatozoa (max/min (LSM)) for two freezing protocols (P5 8C and P15 8Ca) and three extenders (M, B, and BBb) Extender

Protocol

Subjective motile (S.E.M. 3.6)

Total motile (CASA; S.E.M. 5.6)

Linear motile (CASA; S.E.M. 4.3)

M

P5 8C P15 8C

65/54 (58) 65/53 (57)

83/56 (65) 79/59 (69)

49/36 (43) 53/41 (46)

B

P5 8C P15 8C

46/24 (32)* 40/9 (20)

46/16 (33) 51/12 (24)

32/12 (24) 43/8 (19)

BB

P5 8C P15 8C

64/45 (56) 58/39 (50)

83/49 (68) 76/41 (59)

65/31 (51)* 50/28 (39)

a

With the addition of the glycerolized fraction at 5 and 15 8C, respectively. M: milk, B: Bioexcell1 3.2% glycerol, and BB: Bioexcell1 6.4% glycerol. * Denotes a signi®cant difference (P < 0:05) within extender between protocols. b

processed using the P15 8C as compared with P5 8C (10  1:4% versus 5  1:4%; P < 0:01), and in extender M15 when processed using P15 8C compared with P5 8C (10  1:4% versus 4  1:4%; P < 0:01). However, no difference between protocols was seen for extenders M5 or M20 (Table 3). 3.3.2. Experiment 2 The extender  protocol interaction in¯uenced sperm motility when using extenders B and BB, but to a lesser extent in samples processed in extender M (Table 4). Within the extenders generally, the SM was higher (P < 0:05) in samples processed using the P5 8C protocol than in those using P15 8C (48  2:6% versus 42  2:6%). A pairwise comparison showed signi®cant differences (P < 0:05) between protocols only in B (P5 8C: 32  3:6% versus P15 8C: 20  3:6%). The TM values did not differ signi®cantly between protocols. The interaction did not in¯uence the proportions of LM spermatozoa, except within BB (P5 8C: 51  4:3% versus P15 8C: 39  4:3%; P < 0:05). No signi®cant differences in the velocity patterns VSL, VAP, and VCL were found due to the interaction (Table 5). Table 5 Velocity (mm/s) in frozen±thawed ram spermatozoa assessed by CASA (max/min (LSM)) for two freezing protocols (P5 8C and P15 8Ca) and three extenders (M, B, and BBb) Extender

Protocol

Straight-line (VSL) (S.E.M. 4.5)

Average-path (VAP; S.E.M. 4.8)

Circular-line (VCL; S.E.M. 5.0)

M

P5 8C P15 8C

134/146 (115) 118/99 (113)

146/114 (128) 129/111 (124)

186/152 (165) 167/138 (154)

B

P5 8C P15 8C

123/92 (111) 133/114 (120)

133/98 (119) 138/119 (126)

152/116 (137) 159/137 (145)

BB

P5 8C P15 8C

115/89 (104) 112/90 (100)

126/98 (113) 124/100 (110)

150/123 (136) 147/122 (133)

a b

With the addition of the glycerolized fraction at 5 and 15 8C, respectively. M: milk, B: Bioexcell1 3.2% glycerol, and BB: Bioexcell1 6.4% glycerol.

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Table 6 Percentages (max/min (LSM)) of frozen±thawed ram spermatozoa depicting sperm membrane integrity (SYBR14/PI) and phases of capacitation (CTC) for two freezing protocols (P5 8C and P15 8Ca) and three extenders (M, B, and BBb) Extender

Protocol

Membrane integrity (S.E.M. 3.5)

Uncapacitated (S.E.M. 2.8)

Capacitated (S.E.M. 2.3)

Acrosome-reacted (S.E.M. 1.7)

M

P5 8C P15 8C

61/46 (57) 65/47 (58)

53/35 (44) 51/40 (45)

56/40 (49) 52/44 (47)

13/4 (8) 11/5 (8)

B

P5 8C P15 8C

39/28 (34) 44/27 (33)

40/24 (33) 35/22 (29)

62/48 (57) 68/55 (59)

14/6 (10) 17/11 (12)

BB

P5 8C P15 8C

58/40 (50) 58/28 (42)

35/26 (31) 39/18 (31)

55/47 (51) 59/44 (50)

28/13 (19) 24/16 (20)

a b

With the addition of the glycerolized fraction at 5 and 15 8C, respectively. M: milk, B: Bioexcell1 3.2% glycerol, and BB: Bioexcell1 6.4% glycerol.

The interaction did not in¯uence the overall values for spermatozoa with an intact membrane, but the percentages of spermatozoa with an intact membrane differed signi®cantly (P < 0:05) within extender BB (P5 8C: 50  3:5% versus P15 8C: 42  3:5%). None of the categories used for assessing the capacitation status was affected by the interaction between protocol and extender (Table 6). 4. Discussion The results of the present study show that increasing egg yolk concentration higher than 5% in milk extender does not signi®cantly improve sperm characteristics after thawing, except for the percentage of membrane intactness, where the preparation containing 10% egg yolk gave signi®cantly better results. Our results also indicate that Bioexcell1 with a glycerol concentration of 6.4% glycerol yielded similar results as did the control extender used (milk-based, with 5% egg yolk). The addition of f/2 at 15 8C did not signi®cantly improve the sperm quality post-thaw. Conversely, membrane intactness was signi®cantly higher at 5 8C compared to 15 8C and the proportion of acrosome-reacted spermatozoa was lower at 5 8C when using milk-based extenders. Although the differences were not always statistically signi®cant for the sperm characteristics studied, all parameters were better when adding f/2 with glycerol at 5 8C, regardless of the egg yolk (in milk extenders) or glycerol (in Bioexcell1) concentration used. Similar results were obtained in previous studies [19]. Mortimer and Maxwell [27] de®ned the kinematics of hyperactivated ram spermatozoa from a subjectively selected sperm population by depicting a hyperactivation pattern with a decreased linearity and VSL, plus an increased VCL and the amplitude of the LHD as the main components. Furthermore, sperm hyperactivation is a phenomenon associated with capacitation [28]. In the present study, we found that the percentages of capacitated spermatozoa increased with an increase in the egg yolk concentration in extender M as well as in Bioexcell1 containing 3.2% rather than 6.4% glycerol, concomitantly with decreased

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frequencies for the motility patterns (VSL, LM, and SM) evaluated. In our study, the CASA analysis took into account all detected spermatozoa without any kind of selection as in the study by Cummings [28]. Consequently, we did not ®nd any differences in the velocity and motility patterns, but all the values decreased progressively, in absolute ®gures, as the concentration of egg yolk in the extenders was increased from 5 to 20%. The protective effect of egg yolk on sperm membrane integrity is largely recognized in farm animals, but it may also be involved in membrane destabilization, as shown by an increased frequency of acrosome damage with an increase in the concentration of egg yolk [29±31]. Despite this, egg yolk has been ruled out as a capacitating factor in itself [32]. This may partly explain why, in the present study, extender M10 had fewer uncapacitated spermatozoa than did M5, despite the higher percentage of spermatozoa with intact membranes. Capacitation represents a destabilization of the sperm membrane, which submits more readily to spontaneous acrosome exocytosis, thus lowering the half-life of the cell population [16]. This side effect of egg yolk may explain the decreased proportion of ram spermatozoa with intact membranes in the extender with 20% egg yolk found in the present study. Sperm membrane parameters, such as intactness and stability, were better in P5 8C than in P15 8C. This could be explained by the reported effect of glycerol as a promoter of capacitation-like changes [33], or by its toxic effect on sperm metabolism [21]. The frequencies of stable (uncapacitated) and capacitated spermatozoa were almost the same in P5 8C and P15 8C; however, the subpopulations showing acrosome reactions were signi®cantly higher in P15 8C than in P5 8C. All tested sperm attributes seemed to be better when adding f/2 at 5 8C than at 15 8C, except in Experiment 2 for the control (M5) and extender BB, where there was almost no difference between the protocols. Concerns about using egg yolk in semen extenders have been a major issue in discussions in the cattle AI industry in recent years and some studies with relevant substitutes have been conducted to address the effect of extenders [22,25,34,35]. Similar concerns also apply to sheep AI. Apart from sanitary concerns, there is a possibility that milk and egg yolk could alter the chromatin structure of the spermatozoa, and thus reduce sperm quality post-thaw [36]. In this study, we have followed the concept of ``wherever possible, additives of animal origin are to be avoided'' [23], by testing the commercially available extender Bioexcell1, with two different glycerol concentrations (®nal concentration: 3.2 and 6.4%, respectively). Most of the variables yielded better results in Bioexcell1 with a concentration of 6.4% of glycerol (BB) than in Bioexcell1 containing 3.2% glycerol. The latter extender (B) also showed lower values for the sperm quality parameters assessed than did the control (milk extender with 5% egg yolk). According to the kinematics of spermatozoa showing hyperactivation, and the results of the CTC test, the spermatozoa processed in extender B underwent a more stressful treatment than did those in BB, probably due to a too low concentration of glycerol. These ®ndings are also underlined by the lower percentage of membrane integrity found in extender B. The fact that BB achieved similar, or even better, values (considering LM) than did the control (milk extender) indicates that this extender, free from additives of animal origin, may be used as an alternative extender when freezing ram semen. In conclusion, an increase of the egg yolk content above 10% in the milk-based extender did not represent any improvement in the post-thaw sperm quality. Bioexcell1 with a ®nal glycerol concentration of 6.4% may be a good alternative, free from additives of animal

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origin, to the traditional milk extender (supplemented with 5% egg yolk) for freezing ram semen. The addition of glycerol at 15 8C compared to that at 5 8C did not present any improvement in the sperm quality after thawing. However, one must remember that the results presented here are based on in vitro evaluations. Further studies must be performed in order to evaluate whether the extenders and freezing conditions tested herein have any in¯uence on the fertility results after cervical AI under ®eld conditions. Acknowledgements We would like to thank Dr. Decuadro Hansen of IMV at L'Aigle, France, for the preparation of Bioexcell1. The authors received ®nancial support for this work from the Swedish Agency for Research Cooperation with Developing Countries (SAREC) and KoÈttboÈndernas Forskningsprogram, Stockholm, Sweden. Dr. Jorge Gil is on leave of absence from the Veterinary Laboratories ``M.C. Rubino'' (DILAVE), Ministry of Livestock, Agriculture and Fisheries (MGAP), Uruguay. References [1] Salamon S, Maxwell WMC. Frozen storage of ram semen. I. Processing, freezing, thawing and fertility after cervical insemination. Anim Reprod Sci 1995;37:185±249. [2] Evans G, Maxwell WMC. Current status of embryo transfer and arti®cial insemination in small ruminants. In: Proceedings of the Satellite Symposium on Reproduction in Small Ruminants, 14th ICAR Congress, Session 3. Sandnes: Norway; 2000, p. 54±9. [3] El-Alamy MA, Foote RH. Freezability of spermatozoa from Finn and Dorset rams in multiple semen extenders. Anim Reprod Sci 2001;65:245±54. [4] Ollero M, Perez-Pe R, Muino-Blanco T, Cebrian-Perez A. Improvement of ram sperm cryopreservation assessed by sperm quality parameters and heterogeneity analysis. Cryobiology 1998;37:1±12. [5] Holt WV. Basic aspects of frozen storage of semen. Anim Reprod Sci 2000;62:3±22. [6] Holt WV. Fundamental aspects of sperm cryobiology: the importance of species and individual differences. Theriogenology 2000;53:47±58. [7] Curry MR, Kleinhans FW, Watson PF. Measurement of the water permeability of the membranes of boar, ram, and rabbit spermatozoa using concentration-dependent self-quenching of an entrapped ¯uorophore. Cryobiology 2000;41:167±73. [8] SoÈderquist L, Madrid-Bury N, Rodriguez-Martinez H. Assessment of membrane integrity after using different procedures to thaw ram spermatozoa frozen in mini-straws. Theriogenology 1997;48:1115±25. [9] Gil J, SoÈderquist L, Rodriguez-Martinez H. In¯uence of centrifugation and different extenders on postthaw sperm quality of ram semen. Theriogenology 2000;54:93±108. [10] Gil J, Rodriguez-Irazoqui M, SoÈderquist L, Rodriguez-Martinez H. In¯uence of centrifugation or low prefreezing extension rates on the fertility of ram semen after cervical insemination. Theriogenology 2002;57:1781±92. [11] Martin IC, Watson PF. The effects of dilution rate and diluent on the fertility of ram semen. J Reprod Fertil 1973;32:310±1. [12] Salamon S, Maxwell WMC. Storage of ram semen. Anim Reprod Sci 2000;62:77±111. [13] D'Alessandro AG, Martemucci AG, Colonna MA, Bellitti A. Post-thaw survival of ram spermatozoa and fertility after insemination as affected by prefreezing sperm concentration and extender composition. Theriogenology 2001;55:1159±70. [14] Salamon S, Maxwell WMC. Frozen storage of ram semen. II. Causes of low fertility after cervical insemination and methods of improvement. Anim Reprod Sci 1995;38:1±36.

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[15] Maxwell WM, Watson PF. Recent progress in the preservation of ram semen. Anim Reprod Sci 1996;42: 55±65. [16] Watson PF. The causes of reduced fertility with cryopreserved semen. Anim Rep Sci 2000;60:481±92. [17] Fiser PS, Fairfull RW. The effects of rapid cooling (cold shock) of ram semen, photoperiod, and egg yolk in diluents on the survival of spermatozoa before and after freezing. Cryobiology 1986;23:518±24. [18] Colas G. Effect of initial freezing temperature, addition of glycerol and dilution on the survival and fertilizing ability of deep-frozen ram semen. J Reprod Fertil 1975;42:277±85. [19] Fiser PS, Fairfull RW. Combined effects of glycerol concentration, cooling velocity, and osmolality of skim milk diluents on cryopreservation of ram spermatozoa. Theriogenology 1986;25:473±84. [20] Fiser PS, Fairfull RW. The effects of glycerol-related changes on post-thaw motility and acrosomal integrity of ram spermatozoa. Cryobiology 1989;26:64±9. [21] Fahy GM. The relevance of cryoprotectant toxicity to cryobiology. Cryobiology 1986;23:1±13. [22] Bousseau S, Brillard JP, Marquant-Le Guienne B, Guerin B, Camus A, Lechat M. Comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin based diluents. Theriogenology 1998;50:699±706. [23] Thibier M, Guerin B. Hygienic aspects of storage and use of semen for arti®cial insemination. Anim Reprod Sci 2000;62:233±51. [24] Garner D, Johnson LA. Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol Reprod 1995;53:276±84. [25] Gil J, Januskauskas A, HaÊaÊrd MC, HaÊaÊrd MGM, Johannisson A, SoÈderquist L, et al. Functional sperm parameters and fertility of bull semen extended in Biociphos Plus1 and Triladyl1. Reprod Domest Anim 2000;35:69±77. [26] Gillan L, Evans G, Maxwell WM. Capacitation status and fertility of fresh and frozen-thawed ram spermatozoa. Reprod Fertil Dev 1997;9:481±7. [27] Mortimer ST, Maxwell WMC. Kinematic de®nition of ram sperm hyperactivation. Reprod Fertil Dev 1999;11:25±30. [28] Cummings JM. Hyperactivated motility patterns of ram spermatozoa recovered from the oviducts of mated ewes. Gamete Res 1982;6:53±63. [29] Watson PF, Martin IC. The response of ram spermatozoa to preparations of egg yolk in semen diluents during storage at 5 or 196 8C. Aust J Biol Sci 1973;26:927±35. [30] Watson PF, Martin IC. Arti®cial insemination of sheep: the effect of semen diluents containing egg yolk on the fertility of ram semen. Theriogenology 1976;6:559±64. [31] Smith RL, Berndtson WE, Unal MB, Pickett BW. In¯uence of percent egg yolk during cooling and freezing on survival of bovine spermatozoa. J Dairy Sci 1979;62:1297±303. [32] Ijaz A, Hunter AG, Graham EF. Identi®cation of the capacitating agent for bovine sperm in egg yolkTEST semen extender. J Dairy Sci 1989;72:2700±6. [33] Slavik T. Effect of glycerol on the penetrating ability of fresh ram spermatozoa with zona-free hamster eggs. J Reprod Fertil 1987;79:99±103. [34] Hinsch E, Hinsch KD, Boehm JG, Schill WB, Mueller-Schloesser F. Functional parameters and fertilisation success of bovine semen cryopreserved in egg-yolk free and egg-yolk containing extenders. Reprod Domest Anim 1997;32:143±9. [35] Van Wagtendonk-de Leew AM, Haring RM, Kaal-Lansbergen LMTE, Den Daas JHG. Fertility results using bovine semen cryopreserved with extenders based on egg yolk and soy bean extract. Theriogenology 2000;54:57±67. [36] Karabinus DS, Evenson DP, Kaproth MT. Effects of egg yolk-citrate and milk extenders on chromatin structure and viability of cryopreserved bull sperm. J Dairy Sci 1991;74:3836±48.