Neuhauser 2018

Prévia do material em texto

Accepted Manuscript
The effect of four different commercial semen extenders on the motility of stallion
epididymal sperm
Stefanie Neuhauser, Patricia Gösele, Johannes Handler
PII: S0737-0806(17)30523-3
DOI: 10.1016/j.jevs.2017.10.015
Reference: YJEVS 2405
To appear in: Journal of Equine Veterinary Science
Received Date: 7 July 2017
Revised Date: 10 October 2017
Accepted Date: 12 October 2017
Please cite this article as: Neuhauser S, Gösele P, Handler J, The effect of four different commercial
semen extenders on the motility of stallion epididymal sperm, Journal of Equine Veterinary Science
(2017), doi: 10.1016/j.jevs.2017.10.015.
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https://doi.org/10.1016/j.jevs.2017.10.015
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The effect of four different commercial semen extenders on the motility of stallion 
epididymal sperm 
 
Stefanie Neuhauser, Patricia Gösele, Johannes Handler* 
 
Pferdezentrum Bad Saarow, Equine Reproduction Unit, Freie Universität Berlin, Bad Saarow, 
Germany 
 
* Corresponding author: Johannes Handler, Pferdezentrum Bad Saarow, Equine 
Reproduction Unit, Freie Universität Berlin, Silberberg 1, 15526 Bad Saarow, Germany. 
E-mail address: johannes.handler@fu-berlin.de (J. Handler) 
 
Keywords: semen preservation, egg yolk, skim milk, defined milk protein extender, horse, 
equids 
 
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Abstract 
To preserve epididymal sperm, only a limited number of sperm is available, and an 
optimum processing method that is applicable for the majority of stallions is therefore crucial 
for successful preservation. The aim of the present study was to evaluate the effect of four 
different extenders that are commercially available for chilled semen on the motion 
characteristics of stallion epididymal sperm. Sperm were harvested by retrograde flush from 
20 epididymides after the routine castration of 10 stallions. Aliquots of sperm samples were 
diluted with each of the four extenders [(E1) skim milk-based, (E2) containing defined milk 
protein, (E3) containing egg yolk, and (E4) containing caseinate]. Total (TMOT, %) and 
progressive motility (PMOT, %) assessed immediately after sperm harvesting and during 
prolonged storage were highest in extenders with selected milk proteins (E2: 54, 24-79 and 
50, 19-78; E4: 57, 23-82 and 52, 14-80) compared to the skim milk-based extender (E1: 40, 1-
66 and 37, 0-62) and the egg yolk-containing extender (E3: 21, 6-48 and 13, 2-40; median, 
min-max for TMOT and PMOT, stored for 48 h at 4° C, respectively). Motility values were 
similar for extenders E2 and E4 during the entire storage period (P > .05), while extenders E1 
and E3 yielded significantly lower values (P .05). Based on the motility results, we recommend extenders 
containing defined milk protein to process epididymal sperm. 
 
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1. Introduction 
Collection of epididymal sperm can be the last opportunity to preserve the genetic 
material of valuable breeding stallions in case of unexpected death or emergency castration, 
or in endangered species with a risk of extinction [1–3]. Because of its capacity to store 
several billion sperm [4], the cauda epididymidis is a considerable sperm reservoir. 
Harvesting sperm by retrograde flush technique can be easily performed with some training, 
and only a minimal operating expenditure is necessary [1]. 
Similar to ejaculated sperm, epididymal sperm quality can distinctly differ among 
stallions, but in actual cases, testing different extenders to identify the most appropriate one is 
not a reasonable option because only a limited number of sperm is available. An optimum 
processing method using a suitable extender, which is applicable for the majority of stallions, 
is crucial for successful preservation. 
The epididymis plays an important role in sperm maturation and the development of 
motility. During transit through the epididymis, the sperm plasma membrane is remodeled; 
this process is continued during ejaculation (“post-testicular sperm maturation”) and within 
the female genital tract, where sperm become ready to fertilize an oocyte [5–7]. Storage in the 
cauda epididymidis maintains spermatozoa in a quiescent state, and sperm become activated 
by contact with seminal plasma (SP) only during ejaculation [6,8]. However, in stallions, 
sperm of the cauda epididymis are already able to fertilize an oocyte [9], and pregnancies 
have been established [10] even following a prolonged cooled storage of epididymides [11]. 
Sperm environment influences motion characteristics, and SP clearly increases sperm motility 
[12]; moreover, other media beside SP are able to increase epididymal sperm motility [13,14]. 
In a previous study from our laboratory, the extender for chilled semen markedly 
increased the motility of sperm collected by retrograde flush with phosphate-buffered saline 
[14]. Therefore, in the present study, we compared the influence of four different semen 
extenders on the motion characteristics of epididymal sperm. 
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2. Material and Methods 
2.1 Animals 
Twenty testes and epididymides were collected after the routine castration of 10 
healthy stallions aged between 2 and 21 (median 5) years and of different breeds (1 Island 
pony, 2 Trotter horses, 2 Friesian horses, 1 Camargue horse, 1 American quarter horse, 1 
German sporthorse, 2 Hanoverian horses) with gonads in normal position and of normal 
shape. Because of the wide range of the stallions’ ages, the animals were allocated to three 
age groups (2 to 4, n=4; 5 to 10, n=3; and >10 years, n=3). Testicles and epididymides were 
packed in examination gloves or plastic vessels filled with saline to prevent desiccation of 
tissues and sent to the laboratory overnight in a Styrofoam box cooled with ice packs. Direct 
contact between testicles and epididymides, and the ice packs, was prevented to avoid 
freezing injuries. On their arrival in the laboratory, testicular temperature was 12.5° C (6.6-
19.8° C). 
 
2.2 Sperm collection and analysis 
The caudae epididymides were dissected from the testicles at room temperature (20° 
C), and sperm were collected by retrograde flush technique [1] using a pipette tip connected 
to a 20-ml syringe filled with 10 ml Dulbecco’s phosphate-buffered saline (DPBS) solution 
with calcium and magnesium (Bio Whittaker®, Lonza, Verviers, Belgium) plus 10 ml air. 
Sperm concentration was measured using the NucleoCounter® (Chemometec, Allrød, 
Denmark), and samples were diluted to 25 x 106 sperm/ml with four different extenders for 
chilled semen: E1) skim milk-based (BotuSemen, Nidacon, Mölndal, Sweden; pH 6.7-7.0, 
360-380 mOsm/L), E2) containing defined milk proteins (EquiPlus, Minitüb, Tiefenbach, 
Germany; pH 6.8±0.2, 320±20 mOsm/L), E3) containing egg yolk (Gent, Minitüb, 
Tiefenbach, Germany; pH 6.6-6.8, 310-330 mOsm/L), and E4) containing caseinate (INRA 
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96, IMV Technologies, L’Aigle Cedex, France; pH 7.0±0.1, 310±20 mOsm/L).For motility 
analysis, the samples were prewarmed at 37° C for 10 min (AccuBlockTM, Digital Dry Bath, 
Labnet International, Edison, NJ), and a volume of 3.0 µl was poured into a prewarmed 
chamber (Leja Standard Count 4 Chamber Slide 20 micron, Leja Products B.V., Nieuw 
Vennep, The Netherlands) and placed on a heated microscope stage (37° C). In addition to 
motility assessment after sperm harvesting, prolonged storage of sperm samples exposed to 
the different semen extenders was evaluated. Therefore, sperm samples were stored in 
separate tubes for 24 and 48 h at 4° C. 
 
Motility and motion characteristics were assessed computer-assisted (Sperm VisionTM, 
Minitube, Tiefenbach, Germany). Total motility (TMOT, %), progressive motility (PMOT, 
%), curvilinear velocity (VCL, µm/s), and straightness (STR, %; average-path velocity/VCL x 
100) were evaluated as parameters for sperm speed and track trajectory; average lateral head 
displacement (ALH, µm) and beat-cross frequency (BCF, Hz) were assessed to obtain 
information about possible precapacitation or hyperactivating events [15,16]. In our 
laboratory, CASA equipment was mounted with a 0.63x video-adapter on the microscope, 
and the following settings were used: 60 frames per second for the evaluation of 6 fields or 
3,000 cells; spermatozoa were observed between 14 and 80 µm2. The playback facility was 
used for quality control and deletion of signals not correlating with a sperm cell. 
Motion characteristics did not differ between the left and right epididymides. 
Therefore, we combined analysis of the data of left and right epididymides. The possible total 
amount of progressively motile sperm was calculated from the number of sperm collected (N) 
and the progressive motility for each extender used: 
[N left epididymis x progressive motility Extender 1 to 4(%)]+[Nright epididymis x progressive motility 
Extender 1 to 4(%)] = progressively motile sperm harvested per stallion. 
 
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2.3 Statistics 
Data were analyzed by JMP statistic software (SAS Institute Inc., Cary, NC). Because 
values were not normally distributed, the Kruskal-Wallis test for nonparametric values was 
used to compare different groups, and the sperm motility values of the stallions were allocated 
to the defined age groups. The nonparametric pairwise comparison was performed using the 
Wilcoxon test on each pair. All sperm parameters are given as median (min-max) according 
to the ESHRE Andrology Special Interest Group guidelines [17]. A P value 10 years old stallions, 
respectively; P > .05). 
 
3.2 Sperm motion characteristics using different extenders 
Total motility (Fig. 1A), progressive motility (Fig. 1B), and some kinematic values 
(Table 2) differed among the extenders (P .05), which also resulted in the highest motility values at all time 
points compared with the other extenders (Fig. 1; P .05). 
 Time E1 E2 E3 E4 
VCL 
(µm/s) 
H0 131.4a 
(0-152.1) 
145.2b 
(119.8-174.1) 
165.4c 
(118.5-182.0) 
145.7b 
(120.4-173.8) 
H24 147.5a 
(43.4-173.4) 
139.7a 
(115.5-165.6) 
138.3a 
(115.6-161.5) 
141.0a 
(109.7-172.1) 
H48 137.2a 
(71.6-164.2) 
143.4a 
(122.1-160.1) 
137.4a 
(94.6-168.1) 
144.8a 
(105.4-186.8) 
STR (%) H0 76a,b 
(0-85) 
77a 
(70-83) 
71b 
(59-81) 
71b 
(54-80) 
H24 77a 
(52-84) 
71b 
(50-81) 
64c 
(50-75) 
74a,b 
(53-81) 
H48 73a 
(59-82) 
69a 
(54-80) 
64b 
(53-73) 
70a 
(49-81) 
ALH (µm) H0 3.0a 
(0-4.1) 
3.2a 
(2.6-3.8) 
3.9b 
(3.1-4.5) 
3.6c 
(3.1-3.7) 
H24 3.3a 3.3a 3.4a 3.4a 
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(2.1-3.9) (2.9-4.0) (2.8-3.9) (2.8-4.0) 
H48 3.3a 
(1.9-3.7) 
3.5a 
(3.0-4.0) 
3.4a 
(2.8-4.0) 
3.5a 
(2.8-4.3) 
BCF (Hz) H0 30.5a 
(0-37.5) 
37.9b 
(33.1-43.0) 
32.5c 
(27.3-36.7) 
33.6c 
(29.0-39.8) 
H24 32.9a 
(17.8-37.1) 
34.1a 
(26.5-37.1) 
31.1b 
(24.9-35.6) 
31.9a,b 
(27.6-36.4) 
H48 30.1a 
(22.5-37.3) 
32.2a 
(27.3-37.5) 
30.9a 
(27.7-34.4) 
30.6a 
(27.8-35.8) 
 
 
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4. Discussion 
The present studyevaluated motion characteristics of epididymal sperm during cooled 
storage using four different extenders. In epididymal sperm, extenders with defined milk 
protein components produced the best results concerning sperm motility. Hence, the selection 
of the extender is a critical factor for the success rate of the preservation protocol of 
epididymal sperm in terms of the total number of progressive motile sperm obtained. Sperm 
motility is still an important parameter to assess semen quality in stallions [18,19]. Total 
motility, progressive motility, curvilinear velocity, and straightness are the kinematic values 
used to assess the percentages of motile sperm and progressively motile sperm, sperm speed, 
and track trajectory, respectively and therefore, these parameters are used to assess the quality 
of sperm motion characteristics [15]. 
Sperm progressive motility is also a crucial parameter for the calculation of the proper 
semen doses for artificial insemination in mares. According to the recommendation of the 
World Breeding Federation for Sport Horses [20], which is 300 x 106 sperm/dose at the time 
of insemination into the corpus uteri, the following hypothetical number of doses can be 
expected for the different extenders used: 3, 43, 13, and 43 doses on the day of semen 
harvesting as well as 29, 40, 13, and 41 doses after 48 hours of cooled storage for E1, E2, E3, 
and E4, respectively. The present data clearly demonstrate that the calculation of insemination 
doses involves other factors in addition to ejaculated sperm. One factor involved the addition 
of SP after thawing, by which the progressive motility of frozen-thawed epididymal sperm 
was improved [14]. E1 in particular achieved a significant increase in sperm motility after 
cooled storage for 48 h and would, therefore, result in a more reliable number of sperm doses. 
However, the wide variation among extenders clearly indicates that more research is 
necessary to determine comprehensive factors for the calculation of optimal insemination 
doses of epididymal sperm. 
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In epididymal sperm, lower progressive motility can be expected than that for 
ejaculated sperm [21]. However, epididymal sperm seem to be more resistant to cold shock 
and freezing injuries than ejaculated sperm [13,21]. Moreover, a lower percentage of nuclear 
defects and knobbed or damaged acrosomes, and a higher percentage of viable sperm, are 
found in epididymal sperm [12]. Cholesterol efflux from the plasma membrane induced by 
contact with SP may be responsible for the lower tolerance to cold shock in ejaculated sperm 
[22]. 
Interestingly, in ejaculated sperm diluted with milk-based extenders, retention of 5% 
to 20% SP is recommended during storage [23,24,25], but in the present study, good motility 
was also observed in epididymal sperm without any SP. Moreover, using a modified Kenney 
extender supplemented with high potassium Tyrode’s medium in ejaculated sperm, removal 
of SP resulted in highest motility during cooled storage compared with samples containing 
10% or 20% SP [26]. Unfortunately, we were not able to compare ejaculated sperm with the 
epididymal sperm of the stallions in this study. However, no relationship of motility was 
observed between ejaculated and epididymal sperm in earlier studies [12,21]. 
Similar to ejaculated sperm, the motion characteristics of epididymal sperm can vary 
enormously among stallions and preservation protocols. Changes in motion characteristics 
can occur in response to a new medium [14,27] or to a different physical and/or chemical 
composition of microenvironments within the female reproductive tract [28]. The progressive 
motility of epididymal sperm after harvesting with Kenney’s medium ranged from 0% to 40% 
[21]. In the present study, clearly higher values for progressive motility were observed using 
extenders E2 (50, 19-78) and E4 (52, 14-80). These values are in accordance with studies for 
ejaculated sperm [29]. In previous studies [3,13,30], flushing epididymides with semen 
extenders resulted in higher motility parameters and kinematic values; in the present study, 
epididymides were flushed with DPBS and an extender was added subsequently. Therefore, 
an immediate protective effect of an extender during sperm harvesting seems advantageous. 
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Prolonged cooled storage of ejaculated sperm usually causes decreased motility and 
kinematic parameters [19,23,28,30]. In the present study, in contrast to these observations and 
our expectations, TMOT and PMOT did not change (E2, E3, and E4) or even increased (E1) 
during prolonged storage. In addition, ALH increased using E1 and E2. However, prolonged 
exposure to the extenders did not result in hyperactivity of the sperm. In the present study, 
dilution of epididymal sperm with extenders containing milk proteins resulted in motility 
values similar to or even higher at different time points than those values in ejaculated sperm 
[23,29]. In contrast, while the addition of 4% clarified egg yolk increased motility parameters 
of ejaculated sperm [23], the extender containing egg yolk resulted in the lowest motility 
values of epididymal sperm during cooled storage. Different protective effects of extenders 
with or without egg yolk on sperm, due to the ejaculation process and the resulting changes in 
the plasma membrane, may be the cause of these controversial results. 
Defined milk proteins in the semen extender are beneficial for semen preservation 
[29]. Caseinate protects sperm even at low concentrations, and sperm quality parameters are 
similar to those of skim milk extenders after 24 h of cooled storage [32, 33], and hydroxide 
peroxide concentrations remain low during cooled storage of ejaculated sperm [33]. In 
addition, phosphocaseinates in the semen extender improved pregnancy rates when using 
semen stored for 24 h [34]. 
All the stored epididymides were cooled for 24 h before harvesting sperm, because a 
laboratory to preserve epididymal sperm will not necessarily be nearby, as is often the case 
when an unexpected event terminates the breeding career of a stallion. Therefore, cooled 
storage of epididymal sperm and transportation for at least 24 h are necessary, and do not 
negatively affect epididymal sperm quality and fertilizing ability [9,13,30,35]. 
Only healthy stallions were included in the present study; however, disease and 
pharmacological treatment did not influence sperm quality after the washing procedure during 
the preservation process, and a similar sperm number can be expected in stallions suffering 
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from severe acute colic that died after abdominal surgery [3]. We recommend collecting 
sperm of a valuable animal in every case of unexpected injury. Even in stallions 2 years of 
age, there is a chance of obtaining a considerable number of progressively motile sperm. 
 
5. Conclusion 
While extender E1 increased total and progressive motilities during prolonged 
incubation, the egg yolk-containing extender (E3) seemed to induce motility in epididymal 
sperm the least. The extenders containing defined milk fractions (E2 and E4) clearly yielded 
the highest values for most sperm parameters evaluated in the present study. Because motility 
remains a major criterion for successful semen preservation, we recommend using these 
extenders with defined milk-protein fractions during the processing of epididymal sperm. 
However, the effect on the fertilizing capacity of sperm needs to be investigated in vivo. 
 
Acknowledgments 
We thank Anja Kaul, Tanja Kosjak, Christoph Lischer, Dirk Mehnert, Pamela Tobehn, 
and Daniela Vogel for collecting gonads and epididymides after routine castration and 
sending them to Pferdezentrum Bad Saarow. 
 
This research did notreceive any specific grant from funding agencies in the public, 
commercial, or not-for-profit sectors. 
 
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Fig. 1. Total (A) and progressive (B) motility of epididymal sperm diluted with four different 
extenders (E1: BotuSemen, E2: EquiPlus; E3: Gent, E4: INRA 96) after 0, 24, and 48 hours’ 
storage period (H0, H24, and H48, respectively). All samples were prewarmed at 37° C for 10 
min before anaylsis. Similar lowercase letters indicate that there is no difference (P > .05) 
between extenders: # means H0 .05) was observed using extenders 2, 3, and 4. 
 
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Highlights 
• Type of fresh semen extender clearly influences motility of epididymal sperm. 
• Fresh semen extender can increase epididymal sperm motility during cooled storage. 
• Extenders containing defined milk proteins are preferable for epididymal sperm.