A02 060

Conservation of fresh ram spermatozoa at 5°C
in the presence of seminal plasma
A. Morrier1, F. Castonguay1,2, and J. L. Bailey1,3 Centre de Recherche en Biologie de la Reproduction,1Département des sciences animales, Université Laval, Québec City, Québec, Canada G1K 7P4; 2Dairy and Swine Research and Development Centre, Agriculture and Agri-Food Canada, Lennoxville, Québec, Canada J1M 1Z3. Received 2 August 2002, accepted 3 February 2003. Morrier, A., Castonguay, F. and Bailey, J. L. 2003. Conservation of fresh ram spermatozoa at 5°C in the presence of seminal
plasma. Can. J. Anim. Sci. 83: 221–227. Seminal plasma aids sperm transport and contains factors beneficial for sperm function.
In artificial insemination, however, diluting the semen reduces the concentration of seminal plasma. To test the hypothesis that
supplemental seminal plasma in extended ram semen improves conservation at 5°C, we added various concentrations of seminal
plasma to semen during storage, and investigated subsequent sperm function in vitro. Semen was divided into three aliquots,
extended in a commercial diluent (Triladyl) supplemented with 0, 10 or 25% (vol:vol) ovine seminal plasma and cooled to 5°C.
After 8 and 24 h at 5°C, sperm were suspended in a modified synthetic oviduct fluid (SOF-m) at 39°C to mimic the female geni-
tal tract at insemination. Sperm aliquots were assessed for motility and chlortetracycline fluorescence after 0, 4 and 8 h in the SOF-
m. No significant differences were observed due to seminal plasma supplementation during conservation at 5°C or incubation in
SOF-m at 39°C. However, decreased sperm motility and fewer non-capacitated sperm were observed concomitant with an aug-
mentation of capacitated and acrosome-reacted cells during incubation in SOF-m. Therefore, the hypothesis that diluent supple-
mentation with homologous seminal plasma improves ram sperm conservation or subsequent sperm function was not supported.
Key words: Ovine, ram, sperm, motility, viability, chlortetracycline fluorescence, artificial insemination, SOF
Morrier, A., Castonguay, F. et Bailey, J. L. 2003. Conservation des spermatozoïdes frais de bélier à 5 °C en présence de plas-
ma séminal. Can. J. Anim. Sci. 83: 221–227. Le plasma séminal facilite le transport du sperme et renferme des facteurs qui lui
sont bénéfiques. Lors de l'insémination artificielle cependant, en diluant la semence, on réduit aussi la concentration du plasma.
Pour vérifier l'hypothèse qu'un supplément de plasma séminal améliorerait la conservation du sperme de bélier allongé à 5 °C, les
auteurs ont ajouté à ce dernier une quantité variable de plasma séminal pendant le stockage et examiné subséquemment la motil-
ité des spermatozoïdes in vitro. Le sperme a été divisé en trois parties, allongé avec un diluant commercial (Triladyl) auquel on a
ajouté 0, 10 ou 25 % (v:v) de plasma séminal d'ovin puis a été refroidi à 5 °C. Après avoir passé 8 ou 24 heures à cette tempéra-
ture, le sperme a été mis en suspension dans un fluide synthétique modifié imitant celui des trompes de Fallope (SOF-m) à 39 °C,
de manière à reproduire les conditions dans le tractus génital de la femelle à l'insémination. Les auteurs ont évalué la motilité des
spermatozoïdes et la fluorescence à la chlortétracycline des aliquotes de sperme laissés 0, 4 et 8 heures dans le SOF-m. Aucune
variation sensible n'a été relevée à la suite de l'addition de plasma séminal lors de la conservation du sperme à 5 °C ou son incu-
bation dans le SOF-m à 39 °C. Néanmoins, la motilité réduite des spermatozoïdes et le plus petit nombre de spermatozoïdes non
capacités sont cohérents avec la hausse du nombre de cellules capacitées et de cellules avec réaction acrosomique pendant l'incu-
bation dans le SOF-m. On en déduit que la dilution du sperme de bélier avec du plasma séminal homologue n'améliore pas sa con-
servation ni le fonctionnement subséquent des spermatozoïdes.
Mots clés: Ovins, bélier, sperme, motilité, viabilité, fluorescence à la chlortétracycline,
insémination artificielle, fluide synthétique modifié des trompes de Fallope Seminal plasma facilitates sperm transport in the female of epididymal compounds (Setchell et al. 1993) or seminal genital tract [reviewed by Yanagimachi (1994)] and may plasma proteins (Barrios et al. 2000). regulate capacitation by contributing decapacitation factors The specific effects of seminal plasma on sperm during to the sperm (Maxwell and Johnson 1999). However, artifi- semen conservation are controversial, but many recent cial insemination implies the dilution of semen, which reports indicate a positive influence of seminal plasma com- removes certain proteins adsorbed to the sperm surface, and ponents on sperm quality. The inclusion of seminal plasma reduces the concentration of natural antioxidants and othercompounds present in seminal plasma. In fact, it has beensuggested that better diluents for freezing and storage of Abbreviations: AR, for acrosome-reacted sperm with dull
fluorescence over the head; B, capacitated sperm for those
semen would be formulated following the systematic study with fluorescence only along the acrosome; CTC, chlorte-
tracycline; F, non-capacitated sperm for those with uniform
3To whom correspondence should be addressed (e-mail: head fluorescence; SOF-m, synthetic oviduct fluid; VAP,
medium path velocity CANADIAN JOURNAL OF ANIMAL SCIENCE
in the media improved the viability and membrane integrity tion. Therefore, each male was used once for this experi- of boar (Maxwell et al. 1998) and ram sperm (Maxwell et al.
ment (n = 12). Volume was determined and morphology and 1997) subjected to flow cytometric sorting. Seminal plasma subjective motility were visually assessed by a trained can suppress premature capacitation and acrosome reactions andrologist, using phase contrast microscopy (400×) at (Cross 1993), due to the presence of membrane-stabilizing room temperature. Samples with less than 75% motile decapacitation factors (Maxwell and Johnson 1999).
sperm were rejected. Sperm concentration was assessed Furthermore, its proteins are thought to prevent or restore the with a calibrated spectrophotometer (Spectronic 20D, membrane integrity of cold-shocked ram sperm (Barrios et Bausch and Lomb, Markham, ON). al. 2000; Pérez-Pé et al. 2001). Although removal of seminalplasma before freezing increased the viability of ram sperm, Semen Preparation and Conservation
plasma membrane integrity, motility and heterogeneity were Immediately after collection, the semen was divided into enhanced when either whole seminal plasma or a specific three aliquots. Each aliquot was diluted with Triladyl fraction were added to the semen diluent (Ollero et al.
(Minitube Canada, Woodstock, ON), a commercial diluent 1997a,b, 1998). Moreover, Pérez-Pé et al. (2002) reported prepared with 20% egg yolk (from commercial eggs kept that seminal plasma prevented the appearance of a capacita- at 4°C until use), as per manufacturer's directions. Triladyl tion-associated tyrosine phosphoprotein in ram sperm during is composed of Tris [Tris(hydroxymethyl)aminomethane], cold-shock, reinforcing the theory that seminal plasma com- citric acid, fructose and 7% glycerol containing tylosin, ponents stabilize the sperm plasma membrane.
gentamycin, lincomycin and spectinomycin as antibiotics.
Maxwell et al. (1999) demonstrated that the addition of Each aliquot was supplemented with either 0, 10 or 25% seminal plasma to frozen-thawed semen prior to artificial (vol:vol) seminal plasma. Semen was extended at room insemination improved sperm motility, with more uncapac- temperature with Triladyl ± seminal plasma (prewarmed to itated and fewer acrosome-reacted sperm compared to 30°C) to reach a final concentration of 1.6 × 109 sperm mL–1 unsupplemented controls. Furthermore, it was found that the (Baril et al. 1993) as normally conducted at CIOQ. Diluted addition of seminal plasma increased pregnancy rates of semen was placed into straws of 250 µL (IMV, l'Aigle, ewes after cervical insemination with frozen-thawed ram France), sealed with polyvinyl acid, progressively cooled sperm. Therefore, the hypothesis of the present study was to 5°C over 3 h in a transport box (CIOQ), and subse- that the addition of homologous seminal plasma to a com- quently maintained at this temperature. This specially mercial semen diluent improves ram sperm quality during designed box contains the exact proportion of ice and conservation at 5°C. Motility and physiological status water at 30°C in order to reach 5°C in 3 h (approximately assessed by the chlortetracycline (CTC) fluorescence assay 0.14°C min–1); thereafter, the temperature can be main- were used as indicators of sperm quality.
tained at 5°C for 36 h. The same transport box was used forall replicates.
MATERIALS AND METHODS
At 8 and 24 h post-collection, one sealed straw of semen was cut open (with clean scissors) and diluted (about 32- Chemicals and Animal Care Guidelines
fold; to 50 × 106 sperm mL–1) in synthetic oviductal fluid All experiments were conducted as authorized by the uni- (SOF-m; Morrier et al. 2002). The SOF-m was composed versity animal care committee and were conducted in accor- of 108 mM NaCl, 7.2 mM KCl, 1.2 mM KH PO , dance with the guidelines of the Canadian Council on 25 mM NaHCO , 5 mM CaCl , 0.5 mM MgCl , 1.5 mM Animal Care (1993). All products used in this experiment glucose, 3.3 mM sodium lactate, 0.33 mM sodium pyruvate, were purchased from Sigma Chemical Co. (St. Louis, MO) 1 mM glutamine, 20 µM penicillamine, 10 µM hypotaurine, unless otherwise specified.
and 50 mg L–1 gentamycin. This solution was supplement-ed with 20% heat-inactivated estrus sheep serum (the Seminal Plasma Preparation
same lot for all replicates) and adjusted to pH 7.36, One ejaculate from each of 10 rams (two Charollais, two 290 mOsm kg–1.
Polled-Dorset, one Romanov, three Hampshire, and two Sperm quality (motility and CTC assay) was assessed Texel) was pooled and centrifuged (12 000 × g, 15 min, immediately following dilution in SOF-m ("Time t0"). The 22°C). Ejaculates with less than 75% sperm motility were samples diluted in SOF-m were incubated in a humidified rejected. The supernatants were collected and held at –20°C atmosphere of 5% CO in air at 39°C. After 4 and 8 h in the until use (Catt et al. 1997).
SOF-m, sperm quality was re-assessed. Figure 1 shows aschematic description of this protocol.
Semen Collection
Six mature Polled-Dorset, one Hampshire, one Texel, one
Sperm Motility Analysis
Charollais, one Arcott Canadian, and two Arcott Rideau For the assessment of sperm motility, three 2-µL samples of rams aged from 2 to 7 yr were housed at the Centre sperm in SOF-m ( 50 × 106 cells mL–1) were deposited into d'Insémination Ovine du Québec (CIOQ, La Pocatière, a 10-µm Makler counting chamber (Sefi-Medical QC). Rams were under light regime, alternating from long Instruments, Haifa, Israel) placed on the motility analyser (16 h of light) to short days (8 h of light) monthly. Semen (Hamilton Thorn Research, Beverly, MA) as described by was collected twice consecutively with an artificial vagina Morrier et al. (2002). At least 200 cells from five random and the two ejaculates were then pooled for experimenta- fields were recorded. The settings used were the following: MORRIER ET AL. — RAM SPERM AND SEMINAL PLASMA
dure, followed by the Fisher's protected least significant dif-ferences (LSD) test when there were significant variableeffects. The statistical model used for sperm parameters(percentage motile, percentage progressively motile, andpercentage CTC pattern F, B and AR sperm) included theeffect of ram, the volume of seminal plasma added (treat-ment), duration of conservation in the diluent at 5°C, andtime of incubation in SOF-m at 39°C. The two-way interac-tions among these variables were also tested and the resid-ual error was the error term. Significance was judged at theprobability level of P < 0.05. SAS Institute, Inc. (1990) soft-ware was used for the analyses. The initial concentrations of the ejaculates and total motili-ty were 3.67 × 109 ± 1.31 sperm mL–1 and 88 ± 4% (means± SD), respectively, with less than 25% morphologicallyabnormal sperm for each ejaculate. The presence of seminalplasma in the Triladyl diluent did not affect the percentageof total motile sperm (P = 0.5321; Fig. 2) at any of the timepoints studied. However, the percentage of total motilesperm increased very slightly during conservation in diluentat 5°C (83% after 8 h versus 86% after 24 h, with a least sig-nificant difference of 2.4%; P < 0.05). The percentage oftotal motile sperm decreased during subsequent incubation Fig. 1. Outline of experimental protocol.
in SOF-m at 39°C (P < 0.05) at all times (0 to 4 to 8 h). The percentage of progressively motile sperm was not frames acquired, 20; frame rate, 20 Hz; minimum contrast, affected by the seminal plasma treatments at any of the time 10; minimum size, 5; lo/hi size gates, 0.2 to 2.0; lo/hi inten- points studied (P = 0.8932; Fig. 3). However, the conserva- sity gates, 0.2 to 1.8; non motile head size, 5; non motile tion in Triladyl diluent at 5°C increased the percentage of brightness, 63; medium path velocity (VAP) value, 95; low progressively motile sperm (41% after 8 h versus 44% after VAP value, 20; slow cells motile, no; and threshold (STR), 24 h when the data of all SOF time points are combined, 60. Only progressive (proportion of cells moving with a cer- with a least significant difference of 1.7%; P < 0.05). The tain straightness) and total motilities (or motile sperm; pro- percentage of progressively motile sperm was lower during portion of cells moving at or above a minimum speed) were incubation in SOF-m at 39°C (P < 0.05) after 8 h of conser- determined for this study.
vation at 5°C. The percentage of progressively motile spermactually increased after 4 h of incubation in SOF-m at 39°C, Chlortetracycline Fluorescence Assay
then decreased again when the semen was conserved for 24 The CTC fluorescence assay used was conducted as h in Triladyl (46, 49 and 36% for times 0, 4 and 8 h in SOF- described previously (Morrier et al. 2002). In brief, the CTC m, respectively; all times were significantly different with a solution was 5 mM cysteine and 750 µM CTC diluted in a least significant difference of 2.3%). buffer of 20 mM Tris base and 130 mM NaCl (pH 7.8). For The percentage of sperm displaying the non-capacitated the assay, 15 µL CTC solutions were mixed with 10 µL CTC pattern (pattern F) was not affected by the addition of sperm diluted in SOF-m, and fixed with 0.5 µL 12.5% seminal plasma to the Triladyl diluent at any of the time (vol:vol) glutaraldehyde solution prepared in 2.5 M Tris points studied (P = 0.5701, Fig. 4). However, the percent- base. Ten µL of this suspension were placed on a micro- ages of F pattern sperm decreased during conservation in scope slide, covered with a coverslip and evaluated by fluo- Triladyl diluent at 5°C and incubation in SOF-m at 39°C (P rescent microscopy. Slides were prepared in duplicate and < 0.05). The inclusion of seminal plasma did not affect the 200 sperm slide–1 were scored at 400× magnification. The frequency of AR-pattern sperm (P = 0.6027) either.
CTC patterns were scored as described by Gillan et al.
Contrary to the F pattern, however, the percentage of AR (1997) as follows: "F" or non-capacitated sperm for those pattern sperm increased with time in either the Triladyl at with uniform head fluorescence; "B" or capacitated sperm 5°C or SOF-m at 39°C. The percentage of B pattern sperm for those with fluorescence only along the acrosome; and was increased due to conservation Triladyl at 5°C (P < 0.05) "AR" for acrosome-reacted sperm with dull fluorescence and duration of incubation in SOF-m (P < 0.05).
over the head.
In the light of reports by Maxwell et al. (1997, 1999) on the The normality of the data was confirmed. Analysis of vari- benefits of seminal plasma on sperm function, our study uti- ance was performed using the General linear models proce- lized a different, more practical approach: the addition of CANADIAN JOURNAL OF ANIMAL SCIENCE
Fig. 2. Percentages of motile sperm (means ± SE) from ram semen
Fig. 3. Percentages of progressively motile ram sperm (means ±
conserved in a liquid state at 5°C for (a) 8 or (b) 24 h in Triladyl SE) conserved in liquid state at 5°C for (a) 8 or (b) 24 h in Triladyl diluent (TDIL = 8 and TDIL = 24, respectively) containing 0, 10 diluent ( TDIL = 8 and TDIL = 24, respectively) containing 0, 10% or 25% (vol:vol) seminal plasma, and after incubation in SOF-m at or 25% (vol:vol) seminal plasma and after dilution in SOF-m at 39°C, 5% CO for 0, 4 and 8 h (n = 12). Seminal plasma did not 39°C, 5% CO for 0, 4 and 8 h (n = 12). Seminal plasma did not affect the percentage of motile sperm (P = 0.5321). Sperm motili- affect the percentage of progressively motile sperm (P = 0.8932).
ty increased very slightly during 8 and 24 h of storage in Triladyl The percentage of progressively motile sperm differed due to con- diluent at 5°C (P < 0.05). Within time of conservation in Triladyl servation in the Triladyl diluent at 5°C only at 4 h of incubation in (TDIL = 8 or TDIL = 24), motility declined (P < 0.05) with each SOF-m (P < 0.05) (TDIL = 8 compared to TDIL = 24). Within successive duration (0, 4 and 8 h) of incubation in SOF-m at 39°C.
time of conservation in Triladyl (TDIL = 8 or TDIL = 24), motili-ty declined (P < 0.05) with each successive duration (0, 4 and 8 h)of incubation in SOF-m at 39°C.
seminal plasma during dilution for artificial insemination,avoiding semen manipulation post-conservation. We testedthe hypothesis that supplementation of ram semen with plasma is detrimental to sperm motility (de Lamirande and homologous seminal plasma during chilled conservation Gagnon 1984) and viability (García and Graham 1987).
would protect sperm function. It is commonly recommend- Ollero et al. (1997b) suggested that those controversial results ed to use cooled ram semen for insemination within 8 h of could be explained by the presence of two components in ram collection (Baril et al. 1993), due to a marked decline in seminal plasma. A higher molecular weight compound, pre- sperm fertilizing capacity within 24 h (Evans and Maxwell sent in the >10 kDa seminal plasma fraction (Ollero et al.
1987; Maxwell and Salamon 1993). Therefore, we evaluated 1997a) would protect ram sperm; however, a low molecular sperm parameters after 8 h (which is within the recom- weight factor would prevent the adsorption of beneficial sem- mended time frame for artificial insemination) and after inal plasma proteins to the sperm after ejaculation. 24 h (when the first marked drop in sperm function is evident).
Several studies have reported a beneficial effect of semi- Contrary to our hypothesis that seminal plasma improves nal plasma on sperm undergoing flow-cytometric process- ram sperm conservation at 5°C, sperm motility, viability and ing (Ashworth 1994; Catt et al. 1997; Maxwell et al. 1997).
distribution of CTC-patterns were not improved with the However, these studies were performed at high dilution inclusion of 10 or 25% seminal plasma. Although several rates (< 1 × 107 sperm mL–1). At such high dilution levels, studies have provided observations that seminal plasma the cells die rapidly, a phenomenon called the "dilution added to semen would be helpful to sperm (Barrios et al.
effect" [Mann and Lutwak-Mann (1981) in Maxwell and 2000; Graham 1994), others have demonstrated that seminal Johnson (1999)], which particularly affects ram sperm MORRIER ET AL. — RAM SPERM AND SEMINAL PLASMA
Fig. 4. Percentages of sperm (means ± SE) displaying CTC patterns F, B and AR during conservation in a liquid state at 5°C for (a) 8 or (b)
24 h in Triladyl diluent (TDIL = 8 and TDIL = 24, respectively) containing 0, 10% or 25% (vol:vol) seminal plasma, and after dilution in
SOF-m at 39°C, 5% CO for 0, 4 and 8 h (n = 12). The presence of seminal plasma did not affect CTC pattern distribution (P > 0.05). The
overall CTC pattern distribution differed within time of incubation in SOF-m at 39°C, due to duration of conservation in Triladyl diluent at5°C (TDIL = 8 versus TDIL = 24; P < 0.05). *For each CTC pattern, the proportion of sperm differs within time of conservation in Triladyldiluent at 5°C (TDIL = 8 or TDIL = 24), due to incubation in SOF-m at 39°C at all times (0, 4 and 8 h; P < 0.05). [Mann (1984) in Maxwell and Johnson (1999)]. However, In our study, sperm were diluted in a medium rich in pro- in our study, 1.6 × 109 sperm mL–1 were used (a compara- teins (Triladyl is prepared with 20% egg-yolk). The egg tively low dilution rate). Furthermore, Harrison et al. (1982) yolk (contained in the diluent) and the estrous ewe serum suggested that using a protein-depleted medium results in (present in the SOF-m) probably play the same role as serum two factors that can explain the dilution effect: (1) poor albumin, thus possibly explaining why the seminal plasma stimulation of motility and (2) the tendency of live sperm to addition did not improve the physiology of ram sperm. adhere to container surfaces. According to Harrison et al.
Maxwell et al. (1999) reported an improvement in fertili- (1982), serum albumin protects against the dilution effect by ty rates and sperm motility, an increase in the percentage of stimulating sperm motility and preventing sperm adhesion.
uncapacitated sperm, and a concomitant decrease in capaci- CANADIAN JOURNAL OF ANIMAL SCIENCE
tated and acrosome-reacted sperm when seminal plasma Baril, G., Chemineau, P., Cognie, Y., Guérin, Y., Leboeuf, B.,
was added to frozen-thawed ram sperm washed of the Orgeur, P. and Vallet, J.-C. 1993. Manuel de formation pour l'in-
semen extender prior to cervical insemination of ewes (com- sémination artificielle chez les ovins et les caprins. FAO, Rome, pared to a control without seminal plasma). Although that study was interesting, their protocol is impractical for large- Barrios, B., Pérez-Pé, R., Gallego, M., Tato, A., Osada, J.,
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extended only in Dulbecco's phosphate-buffered solution Bedford, S. J., Jasko, D. J., Graham, J. K., Amann, R. P.,
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