Effects of seminal plasma and three extenders on canine semen stored at 4 °C

EFFECTS

OF SEMINAL ON CANINE

PLASMA SEMEN

AND THREE EXTENDERS STORED AT 4°C

A. Rota, B. Strom and C. Linde-Forsberg

I

Department of Obstetrics and Gynecology Faculty of Veterinary Medicine Swedish University of Agricultural Sciences Box 7039,750 07 Uppsala, Sweden Received

for publication: Accepted:

December Apr+I

16, 1994

18, 199s

ABSTRACT Semen preservation and artificial insemination (AI) in the canine has become a common practice in veterinary medicine. Chilled dog semen is easy to handle, and several extenders can be used. The aim of this study was to compare the effects on canine spermatozoa of seminal plasma and 3 extenders commonly used for chilled semen preservation in clinical practice. The characteristics evaluated were sperm motility; velocity; plasma membrane status (assessed with a fluorescence staining technique and hypo-osmotic swelling test); acrosome morphology; semen pH; and semen osmolarity. These criteria were monitored daily in the ejaculates of 11 dogs. The ejaculates were divided into 4 aliquots. Each aliquot was extended in autologous seminal plasma, egg-yolk Tris, egg-yolk milk or egg-yolk cream and preserved at 4°C for 4 d. In 10 of 11 semen samples extended in autologous seminal plasma, motility had already decreased to 0% by Day 2, and the percentage of spermatozoa with intact membranes was lower than in the 3 extenders (PO.O5). Egg-yolk Tris extender seems to be superior to the other extenders tested, to preserve dog semen at 4°C although differences were not significant for all the parameters. Key words: dog, chilled semen, extended

semen, HOS-test,

fluorescence

Acknowledgments This study was supported in part by grants from The Thure F. and Karin Forsberg Foundation, and The Swedish Kennel Club. We also gratefully acknowledge the encouragement by the Faculty of Veterinary Medicine of the University of Pisa, especially The Department of Clinical Surgery and Obstetrics. 1 Author to whom reprint requests should be adressed.

Theriogenology 44:665-900. 1995 0 1995 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

0093-691 X/95/$1 0.00 SSDI 0093-691X(95)00276-2

886

Theriogenology INTRODUCTION

The use of chilled extended semen for artificial insemination (AI) in the canine has become increasingly popular over the last 2 decades. Different types of semen extenders have been evaluted for their capability to keep chilled dog semen motile over time (9,16,33). These extenders included citrate, Tris, phosphate or glycine buffers, heated or dried milk, sterilized cream, with or without the addition of sugars, egg yolk and antibiotics. In a field study comprising 269 AIs with chilled semen preserved undiluted or in different extenders, the best results were obtained with egg-yolk Tris (62.5%), Tris (57.1%) and egg-yolk cream (51.1%) extenders. However, due to the small number of AIs using egg-yolk Tris and Tris, the results were not conclusive (26). Chilled extended dog semen is usually used within 24 h when transported within the country or between neighboring countries. If spermatozoa were to retain not only their motility but also their fertilizing ability for 2 to 3 d, chilled extended dog semen could be transported over longer distances. Compared with the use of chilled extended semen, the freezing and shipping of and AI with frozen canine semen requires more sophisticated techniques and equipment, and pregnancy rates generally are lower (14,18,25,27). Intrauterine deposition of frozen-thawed semen gives a higher pregnancy rate and larger litter size than intravaginal deposition (5,15). These differences may be due to the reduced capacity of fiozenthawed spermatozoa to pass through the cervix. Uterine deposition of semen can be achieved surgically (4,36), which entails some stress and surgical risks for the bitch, or transcervically with the Norwegian catheter (5) or with the aid of an endoscope (7,39). With chilled extended semen, the pregnancy rate remains acceptable also after vaginal deposition of semen. The best way of evaluating extenders for semen preservation is to compare conception rates after AI. However, this is usually not possible under experimental conditions with dogs; therefore, assessment of sperm characteristics important for fertilizing ability are evaluated in the laboratory. By combining various tests a significant correlation with fertility can be obtained (3). Spermatozoa must have good motility in order to reach the oviduct and penetrate the investments around the oocyte. An intact plasmalemma is a prerequisite for maintaining membrane potential, ionic microenvironment and pH. Furthermore, spermatozoa cannot survive in the female reproductive tract and bind to the zona pellucida and the vitelline membrane without having an intact plasmalemma Damage to the acrosome can also result in infertile spermatozoa, since acrosomal enzymes are needed for penetration of the zona pellucida and fusion with the oocyte (8735). The aim of this study was to compare 3 commonly used extenders and seminal plasma in terms of their effects on canine spermatozoa, as judged by characteristics such as motility and the integrity of the plasma membrane and acrosome. MATERIALS AND METHODS

Semen was obtained from 11 privately owned dogs: 2 German shepherds, 2 Bernese mountain dogs, 1 Flat-coated retriever, 1 Labrador retriever, 1 Rottweiler, 1 Hovawart and 3 crossbreeds.

887 Their ages ranged from 1.5 to 8 y, and 3 of the dogs were known to be fertile. Semen Processing One ejaculate from each dog was collected in a calibrated plastic vial by digital manipulation (25). Semen collection was discontinued when the third fraction appeared in order to minimize the amount of prostatic fluid in the sample. The volume of each collection was determined, and the sperm concentration was measured with a photometer (SpermaCue, Leo Diagnostics Inc., Helsingborg, Sweden). Aliquots of freshly ejaculated semen were then separated for evaluation prior to any other treatment. The remaining semen was centrifuged at 700 g for 6 min, and the supematant was removed. To obtain equal sperm concentration in all extended samples from each single semen collection, the remaining pellet was resuspended to 0.8 ml in the supematant and divided into 4 parts. Bach part was mixed with each of the different extenders or with a corresponding volume of autologous seminal plasma. The sperm concentration in the extended semen samples varied from 30 to 105 x 106/ml between dogs. Extended semen was stored at 4°C and evaluated daily from Day 0 (day of collection, timed immediately after mixing pellet with extender) to Day 4. Semen Extenders The following extenders were used: 1) Autologous seminal plasma, obtained as the supematant after semen centrifngation. No egg yolk or antibiotics were added. 2) Egg-yolk Tris extender: 3.025 g Tris(hydroxymethyl)aminomethane, 1.7 g citric acid, 1.25 g fructose, and distilled water added up to 100 ml. Egg yolk (20%) and antibiotics (benzylpenicillin 1 mg/ml and dihydrostreptomycin sulphate 1 mg/ml) were also added. 3) Egg-yolk milk extender: pasteurized milk with 0.1% fat. Egg yolk and antibiotics were added as described for the egg-yolk Tris extender. 4) Egg-yolk cream extender: ultra-high-temperature-treated cream containing 12% fat; egg yolk and antibiotics were added as described above. The Egg-yolk Tris extender was made up as a single batch and was stored frozen together with the pasteurized milk. Egg yolk and antibiotics were added immediately before use. Semen Evaluation Before the extenders were added, semen smears for sperm morphology analyses and differential cell counts were made using a drop of fresh semen from each ejaculate, and an aliquot was fixed in 10% formol-saline. Sperm morphology was assessed under a phase contrast microscope for samples fixed in buffered formol saline (6) and under a light microscope in carbol fuchsine-stained smears (38). Dry smears were also stained according to Papanicolaou to detect for the presence of cells other than spermatozoa (32). The following tests were performed on fresh semen on Day 0 and on extended semen on Days 0 to 4:

888

Theriogenology

Acrosome morphology was examined by counting 100 spermatozoa in a dry smear after staining with Sperma@ stain (Stain Enterprises, P.O. Box 152, Wellington, South Africa). The spermatozoa were classified into 3 groups according to the appearance of their acrosomes: Group A were normal appearing acrosomes; Group B were vesiculated acrosomes, swollen acrosomes and acrosomal losses; and Group C were other acrosomal abnormalities (3 1). Motility was assessed in S-p1 aliquots and examined at 37°C in a Makler chamber under a phase contrast microscope. At least 3 fields from each sample were videotaped. Sperm velocity, using a scale from 0 (absence of any movement) to 5 (strong, vigorous forward movement) and the percentage of motile spermatozoa were subjectively assessed from tape recordings by 2 operators. Both the pH and osmolarity were measured and recorded for each extender prior to use. The measurements were made on Day 0 for fresh semen and daily for the extended semen. Sperm membrane integrity was assessed using a fluorescent staining technique (Harrison and Vickers 1990, modified; 19,29) and the hypo-osmotic swelling (HOS) test (23). A staining media for the fluorescent probes was prepared within 1 h prior to use, using 20 pl of formaldehyde (2.5 mg/ml in H20), 20 pl of 6-carboxyfluorescein diacetate (C-FDA; Calbiochem, Sweden) and 20 pl of propidium iodide (PI; Sigma Chemical Company Ltd, St. Louis, MO, USA) for each ml of saline medium (140 mM NaCl, 1OmM glucose, 2SmM K+, 0.5 mg polyvinyl alcohol/ml, 0.5 mg polyvinyl pyrrolidone PVP/ml and 20 ml Hepes, pH 7.5, 300 mosmikg). Semen (0.1 ml) was diluted with 0.3 ml of staining medium and incubated in the dark at 30 “C for 15 min. A 5-pl aliquot of stained suspension was placed on a slide and covered with a coverslip, and the fields, well dispersed over the slide, were observed at x 400 magnification with epifluorescence UV-illumination on a Diaplan Leitz microscope. Staining with C-FDA was assessed using a H3-13 filter block (Leitz 513673-719, blue excitation range, with a BP420-490 excitation filter, a RKP510 dichromatic mirror and a LP520 suppressor filter), while staining with PI was assessed using an N2 filter block (Leitz 513609, green excitation range, with a 530560 excitation filter, a RKP580 dichromatic mirror and a LP580 suppressor filter). For each sample stained with C-FDA/PI 100 cells were counted on 2 different slides (total=200) and classified as Group A, having an intact plasmalemma when stained green with C-FDA and unstained with PI; Group B, having a damaged plasmalemma but an intact acrosome when the acrosome stained green with C-FDA but the post-acrosomal region stained red with PI; and Group C, having both a damaged plasmalemma and damaged acrosomal membrane when the cells were unstained with C-FDA but stained red with PI (29). To assess plasmalemma integrity with the HOS-test, a 60-mOsm fructose solution was prepared, stored at -18°C and thawed prior to use. In a test tube 0.1 ml of semen (fresh or extended) was added to 1 ml of the solution and incubated for 45 min at 37°C. After incubation 1 drop was placed on a slide, covered with a coverslip and examined under a phase contrast microscope (x 400). A total of 200 sperm cells was counted, and the percentage of curled/swollen spermatozoa (i.e., with an intact plasma membrane) was determined (23).

889

Theriogenology Statistical Analysis

The extenders were compared using the Wilcoxon matched pairs signed rank sum test, adjusting the P-values using the Bonferroni method (2). A comparison was also performed over days within the extenders. Values were considered to be statistically significant when P < 0.05. The analysis was performed using Minitab statistical software (Minitab Inc., State College, PA, USA). RESULTS Semen Quality Fresh semen characteristics varied between dogs and are summarized below (Table 1).

Table 1. Characteristics of fresh semen (n = 11 dogs) Mea&SD Total

number

of spermatozoa

Motility

(%)

Velocity

(scored

4.9+0.3

Intact

plasmalemma

(HOS-test)

plasmalemma

(C-FDA/PI)

(mOsm)

Acrosomes:

vesiculated

Acrosomes:

other abnormalities

Tail defects

(%) (%)

93.6+3.7 76.8f11.5 6.3f0.3

PH Osmolarity

Normal

916+348 78.6f13.6

O-5)

Intact

Pathological

(x106)

heads

315.9k21.8 /swollen

/ lost (%) (%)

(%)

2.7f2.0 5.9k2.6

(%)

appearing

10.7k8.3

17.4f22.0 spermatozoa

(%)

63.7f26.9

Motility Mean sperm motility on Days 0 to 4 is shown in Table 2 and Figure 1. On Day 0, immediately after extending the samples, no significant difference in mean motility was seen. On Day 1, the mean motility in autologous seminal plasma was significantly lower than corresponding means in all the extenders. No significant difference was observed among the 3 extenders on Day 1. For Days 2 to 4, mean motility in autologous seminal plasma was significantly lower than the means in egg-yolk Tris and egg-yolk milk. Mean motility in egg-yolk Tris was significantly higher than the means in egg-yolk milk and egg-yolk cream for Days 2 to 3 and significantly higher than the mean in egg-yolk cream for Day 4.

890

Theriogenology

Table 2. Motility and velocity (Mean + SD) of the semen preserved in autologous seminal plasma and in the 3 extenders from Day 0 to Day 4 (n = 11 dogs). Day 1

Day 2

Day 3

67.1i22.1

21.6Q9.7

1.9k2.5

o.oti.0

4.521.0

2.4k1.5

0.6iO.8

o.o~oo.o

o.oti.0

76.8+14.0

73.6Q5.5

7o.ti15.4

60.9k18.9

53.e20.1

4.7kO.5

5.oxl.o

4.8kO.4

4.5io.5

4.23.9

75.0214.5

57.7Q4.5

48.2k17.2

35.9218.7

3o.ti19.4

5.0?0.0

4.4kO.6

3.9kO.9

3.520.9

77.3k13.3

60.9224.7

48.e28.5

29.1ti9.7

14.1*20.8

4.820.4

3.9kl.l

3.5i1.6

2.2k1.7

1.7t1.5

Day 0 ASP motility velocity EYT motility velocity

EYM motility velocity EYC motility velocity ASP= autologous

seminal

Day 4 O.MO.0

2.7tl .O

plasma;EYT= egg-yolk Tris; EYM= egg-yolk milk; EYC= egg-yolk cream.

r

1 70 60 50 40 30 20 10 0 ASP

Figure 1. Mean motility in the semen preserved in autologous seminal plasma (ASP) and in the 3 extenders from Day 0 to Day 4 (n = 11 dogs). EYT= egg-yolk Tris; EYM= eggyolk milk; EYC= egg-yolk cream. The mean sperm motility in the egg-yolk Tris extender did not decrease significantly from Day 0 to Day 1, whereas this decrease was significant in the autologous seminal plasma, eggyolk milk and egg-yolk cream. From Day 2 onwards mean motility was significantly lower in both autologous seminal plasma and the 3 extenders compared with that of Day 0.

Theriogenology

891

100 90

-+-Dog 1 -q-Dog 2

80 70

-s--Dog3 -e-w4 -A-D%5 --+-Dog6

60 $0 2 40

--a-m%7 -o--W8

30

~

20

-&-Dog9 ~ -+-Dog lOi

10

-o-Dog

1’ /

0 0

1

2

3

4

Day

Figure 2. Motility of the semen preserved at 4°C for 4 days in autologous seminal plasma.

90

80 -,+Dog 60 ,x $ 50 r

40 30

i

!

pi

-+Dog6

,

-m-Dog7 -o-Dog8

;~ in

-Dog9

20 :

3

-.-Dog4 -Dog5

+Dog -Dog

10

10 11

0 0

1

2

3

4

Day

Figure 3. Motility of the semen preserved at 4°C for 4 days in egg-yolk cream.

In autologous seminal plasma, motility in the individual dogs dropped drastically from one day to another throughout the period of study: in 10 of 11 semen samples motility was 0% by Day 2 (Figure 2). Reductions in motility were also pronounced in the egg-yolk cream extender;

892

Theriogenology

100

1

90

-+-Dog

1

+-Dog

2 3

60 ,x s 50

-_x-Dog -o-b4 -A-w5 +-Dog6

E 40

-m--w

7

80 70

20

-o--Dog8 j -it-J%9

10

-o-mz

30

-+w+

10 11

0 0

1

2

1

4

3

Day

Figure 4. Motility of the semen preserved at 4°C for 4 days in egg-yolk milk.

100 90

-+-Dog 1 -_x-Dog 2 __x-Dog 3 +mit4

I--

80 70 60

-A-m25

-+Dog6 -m-Dog 7 -o-D%8

30

*-Dog9 +L%31 -o-m1

20 10 0 0

1

2 &Y

3

4

Figure 5. Motility of the semen preserved at 4’C for 4 days in egg-yolk Tris.

in 2 of the dogs it dropped to 0% on Day 1 and Day 2 respectively (Figure 3). By contrast, in the remaining 2 extenders (Figures 4 and 5) no drastic drops were seen, and motile spermatozoa were present until Day 4 in all the samples. Between-dog variation in motility was greater in egg-yolk milk and egg-yolk cream extenders than in egg-yolk Tris extender.

Theriogenology

893

In egg-yolk Tris extender the mean score for sperm velocity remained higher than Grade 4 throughout the 4 days, whereas it had dropped below 4 by Day 1 for autologous seminal plasma and egg-yolk cream and by Day 2 for egg-yolk milk extenders (Table 2). Membrane Integrity Integrity of the plasma membrane for the semen preserved in autologous seminal plasma and as swollen spermatozoa and as spermatozoa retaining C-FDA (green fluorescence), is shown in Table 3. in the 3 extenders, determined

Table 3. Percent of intact membranes (Mean + SD) estimated with the hypoosmotic swelling (HOS) test and C-FDA/PI staining (green fluorescence) for spermatozoa preserved in autologous seminal plasma and in three extenders from Day 0 to Day 4 (n = 11 dogs).

HOS-test

c-FDA/PI

Day 0

Day 1

Day 2

Day 3

Day 4

Seminal plasma

93.W.2

85.3i5.8

67.9Y6.0

55.9X21.5

46.2L23.9

Egg yolk Tris

91.24.1

91.324.2

87.8t4.4

86Z5.3

86.W.4

Egg yolk milk

92.OL3.3

90.9L2.6

88.2L4.4

85.9%5.3

81.4?5.0

Egg yolk cream

92.E4.2

89.333.1

88.13.8

84.13.0

83.W.3

Seminal plasma

71.ti13.2

53.5k14.4

42.4216.7

26.E19.3

16.4*11.1

Egg yolk Tris

83.ti4.9

80.2%.4

78.4F6.4

70.327.9

72.1+10.4

Egg yolk milk

86.9L5.9

80.73.3

79.213.3

79.e15.7

81.35.2

Egg yolk cream

87.ti4.7

80.8k7.3

78LX7.8

78.912.9

76.W.7

C-FDA/PI= 6-carboxyfluorescein

diacetate

The HOS-test revealed that the percentage of spermatozoa with damaged plasma membranes was higher in autologous seminal plasma than in any of the 3 extenders, the difference being significant on Days 2 to 4. No significant difference was found when comparing the 3 egg-yolk extenders. The decrease over time in the proportion of spermatozoa with intact membranes had already become significant by Day 1 in autologous seminal plasma, whereas in the extenders the decrease had become significant by Day 2 (egg-yolk Tris and egg-yolk milk) or Day 3 (egg-yolk cream). The fluorescence C-FDA/PI staining technique showed that by Day 1 the percentage of spermatozoa with intact membranes was already significantly lower in autologous seminal plasma than in semen extended in egg-yolk Tris, egg-yolk milk or egg-yolk cream. A decrease in

894

Theriogenology

the number of spermatozoa with intact membranes had already become significant by Day 1, whereas in egg-yolk Tris and egg-yolk cream the decrease first became significant on Day 2. The decrease in membrane integrity in the egg-yolk milk extender had still not became significant after 4 d. Results from the HOS-test and the fluorescence C-FDA/PI staining technique were not correlated on Day 0, whereas the correlation coefficients on Days 1 to 4 were 0.484, 0.664, 0.733 and 0.830, respectively. Acrosome Staining Acrosomal abnormalities thought to represent secondary changes, such as different stages of the “false” acrosome reaction, or acrosome degeneration, i.e., vesiculated acrosomes, swollen acrosomes and loss of acrosomes (3 l), were grouped together (Croup B). The mean percentages of acrosomes classified as belonging to Croup B throughout the 4-d period for the 3 extenders and for autologous seminal plasma are shown in Figure 6. In this group the mean of acrosomal changes was significantly lower for egg-yolk Tris than for autologous seminal plasma on Days 3 and 4. Other differences between the extenders were not significant. Spermatozoa in autologous seminal plasma and in egg-yolk milk and egg-yolk cream extenders showed a significant increase over time in acrosome changes, which was not observed in egg-yolk Tris extender.

1

I

Figure 6. Mean combined percent of vesiculated acrosomes, swollen acrosomes and loss of acrosomes in the semen preserved in autologous seminal plasma and in 3 extenders from Day 0 to Day 4 (n = 11 dogs). ASP= autologous seminal plasma; EYT= eggyolk Tris; EYM= egg-yolk milk; EYC= egg-yolk cream.

a95

Theriogenology

The percentage of spermatozoa showing other acrosomal abnormalities, thought to be primary defects (Group C), did not change significantly over the 4-d period, nor did it differ significantly between extenders. PH The mean pH recorded for the 3 extenders prior to semen dilution was 6.68 (iO.04) for eggyolk Tris, 6.56 (kO.04) for egg-yolk milk and 6.40 (kO.02) for egg-yolk cream. For semen diluted in autologous seminal plasma the pH had increased significantly by Days 3 to 4, while for semen extended in egg-yolk cream, egg-yolk Tris and egg-yolk milk the pH had decreased significantly by Days 2 to 4, Days 3 to 4 and Day 4, respectively. Osmolarity The osmolarity of the extenders before adding semen was 320.7 (k28.3) mOsm for egg-yolk Tris, 297 (k3.35) mOsm for egg-yolk milk and 308 (k4.3) mOsm for egg-yolk cream. Osmolarity increased in all extended semen samples on Day 1, declined on Day 2 and then increased again. The changes were significant for the egg-yolk cream extender on Days 1,2 and 4. DISCUSSION To test the suitability of extenders for fresh chilled canine semen it is necessary to study parameters such as sperm motility, sperm membrane status and acrosomal integrity after prolonged storage. By combining 2 or more of the above tests a significant correlation with fertility should be obtained. The extenders used in this study were chosen because they are widely used by practitioners, as they are easy to prepare. We also chose to include autologous seminal plasma in the study, since semen is sometimes sent unextended, and to use it as a control. Antibiotics were not added to autologous seminal plasma because this is not normally done when unextended chilled dog semen is used in clinical practice. Several tests were performed to more completely characterize the morphological and functional aspects of the spermatozoa during preservation. In most previous studies on chilled dog semen only motility was assessed. Kumi-Diaka and Kumi-Diaka and Badtram (23,24) used a combination of assessment tests (motility, hypoosmotic swelling, acrosome reaction) on chilled dog semen, but they tested only one extender. The dogs in our study were not selected for semen quality; thus they varied in their individual semen characteristics, as is the case in clinical practice. Motility was estimated subjectively. Although computerized, objective techniques are available, they could not be used for the milk- and cream-based extenders. This was compensated for by having each sample evaluated by 2 observers. Several other extenders have been evaluated for their ability to maintain motility for varying periods of time (up to 16 d). Morton and Bruce (28) compared the motility of canine semen diluted in a Tris-based extender with that of

896

Theriogenology

undiluted semen, both preserved at 4’C for 5 d. Motility was still very high after 4 d in the extended semen, whereas the undiluted samples had a reduced ability to survive at 4°C. In our study, semen diluted in autologous seminal plasma already showed poor motility on Day 1. The short survival of canine spermatozoa in seminal plasma has been previously reported ( 12). In the present study we cannot say whether seminal plasma was actually detrimental to sperm survival. There is little doubt, however, that the addition of extenders was beneficial and significantly improved sperm longevity. From Day 2 of storage at 4°C motility was preserved better in eggyolk Tris than in the other extenders. The significance of this finding for sperm fertilizing capacity remains to be studied. Plasma membrane integrity was evaluated using 2 techniques. The HOS-test is a simple, quick and inexpensive method to assess functional integrity of the plasma membrane (13,23). Exposure of spermatozoa to a hypo-osmotic solution causes an influx of water through the plasmalemma until an osmotic equilibrium is reached, resulting in a swollen cell, mainly manifesting itself as a curled sperm tail. Swelling indicates that the transport of water across the plasma membrane has occurred normally (21). The C-FDA/PI staining technique, derived from flow cytometric analysis, is a more complex technique, and it has been previously used for ram and boar spermatozoa (19,29). The C-FDA penetrates the plasma membrane and is subsequently de-esterified inside the cell. The resultant free carboxyfluorescein, green fluorescent and impermeable, accumulates in the cytoplasm of intact spermatozoa. The PI cannot penetrate the plasma membrane; thus it only enters and stains cells with a damaged (permeable) plasma membrane, binding to cellular DNA and emitting a red fluorescence. With the fluorescence technique, the percentage of intact cells decreased significantly over time in all extenders except in egg-yolk milk. With the HOS-test, the percentage of swollen cells (i.e., with an intact membrane) decreased over time in all extenders, the integrity of the plasma membrane being slightly but not significantly better in the egg-yolk Tris extender. The percentage of spermatozoa classified as having an intact plasma membrane was always higher when using the HOS-test than C-FDA/PI staining. This may be due to an independent function between the plasma membrane in the sperm head and in the sperm tail. Differences in the composition and stability of the plasma membrane between regions of the sperm cell have been previously described (22). The plasma membrane overlying the sperm head is considerably more labile than that of the flagellum. This was shown in ram spermatozoa after a freezing-thawing process (20). It has been proposed that the sperm head and tail might be metabolically as well as structurally isolated from each other by the posterior ring (22). This anchorage of the plasma membrane to the caudal part of the head of the spermatozoon could allow the plasma membrane apposed to the tail region to swell regardless of any minor damage to the head region. The presence of swelling in the head region was not evaluated in our study, but, using electron microscopy, it has been shown in dog spermatozoa subjected to the HOS-test (13). Jeyendran et al. (21) argued that an intact plasma membrane was presumably also present on the head of the spermatozoon when the HOS-test was positive, since they found a good correlation between swelling of human sperm tails and the ability of those spermatozoa to undergo capacitation and penetrate oocytes, which requires a functioning plasma membrane in the head region. In our study, no correlation between the percentage of spermatozoa taking on a green fluorescence after C-FDA/PI staining and the percentage that swelled in a hypoosmotic solution was found on Day

Theriogenology

a97

0, but a good correlation was found on the following days. This initial discrepancy could have been due to the presence of minor damage to the head region of the spermatozoa (more sensitive), which can be detected by fluorescence staining but not by the HOS-test. In previous studies the effects of chilling and storing canine semen on HOS test results were assessed (23,24). Preserving canine semen in an egg-yolk Tris-based extender for 24 h did not damage the plasma membrane (24), similar to our findings. With both the HOS-test and FDA/PI the most pronounced changes in the plasma membrane were found in the spermatozoa preserved in seminal plasma. A detrimental effect on the spermatozoa1 plasma membrane caused by cationic peptides present in seminal plasma has been reported in the bovine, and cold shock itself increases membrane permeability. Egg yolk cationic lipoproteins have been found effective in protecting from cold shock and injury induced by these seminal plasma components in the bull and ram semen during dilution, ambient temperature storage, cooling and freezing, since these bind to the sperm plasma membrane (17,34,37). Although we cannot say whether seminal plasma was detrimental, the addition of extenders was beneficial and significantly improved the percentage of intact plasma membranes in our study. Oettle (30) reported an increase in acrosome alterations in canine spermatozoa after dilution and chilling (prior to freezing). A high percentage of swelling of the apical ridge of acrosomes of bull spermatozoa preserved at 4°C was found when stored in seminal plasma for 1 to 5 d. Swelling was inhibited by replacing the seminal plasma with egg-yolk-citrate (1). In our study, the largest increase in acrosome modifications was found in the semen stored in autologous seminal plasma. Egg-yolk Tris maintained the integrity of acrosomes better than egg-yolk milk and egg-yolk cream extenders did, possibly because of its capability to maintain the spermatozoa alive and motile longer, thereby preventing the “false” acrosome reaction that occurs in association with sperm death or irreversible damage (1 ,10,35). The mean pH and osmolarity found in fresh semen (first and second fraction) were similar to those of previous reports (11). During storage the pH in egg-yolk Tris, egg-yolk milk and eggyolk cream decreased only slightly, reflecting the good buffering capacity of the 3 extenders (although changes were more pronunced in egg-yolk cream than in egg-yolk Tris and egg-yolk milk). There was a pH increase in autologous seminal plasma, possibly due to the production of spermatozoa1 or, because no antibiotics were added in these samples, bacterial metabolites. The changes in osmolarity during the study were always within the physiological range. Spermatozoa1 velocity and motility after 4 d of storage were highest in egg-yolk Tris (although motility was not significantly higher than in egg-yolk milk on Day 4), and variability between dogs was lower with this extender. On Day 4, neither membrane status nor the number of vesiculated, swollen and lost acrosomes (Group B) differed significantly between any of the 3 extenders. However, the frequency of Group-B acrosomes was significantly higher in autologous seminal plasma than in egg-yolk Tris. After 2 d of storage, which is usually the maximal time needed for transport, no differences were found between the egg yolk extenders when evaluating plasma membranes and acrosomes; however, motility and sperm velocity were preserved best by egg-yolk Tris.

898

Theriogenology

Autologous seminal plasma was shown to be unsuitable for preserving canine semen at 4°C: motility, velocity and percentage of intact membranes (fluorescence staining) were already significantly lower than in the 3 egg yolk extenders by Day 1. Egg-yolk Tris showed the best ability to maintain sperm characteristics for the first 24 h during which no significant changes were found. Furthermore, although no significant differences among the 3 extenders were observed for the other parameters, sperm motility and velocity over the study period were maintained best in egg-yolk Tris. Moreover, less variation in motility between dogs was observed with egg-yolk Tris extender. Thus, for the preservation of dog semen at 4°C egg-yolk Tris seems to be superior to the other extenders tested, although further studies on the fertility of spermatozoa preserved under these conditions are needed.

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