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Comparison of the Effects of Four Freezing Methods on Motility Characteristics, Morphology, and Viability of Postthaw Stallion Epididymal Sperm
Journal of Equine Veterinary Science xx (2014) 1–7
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Original Research
Comparison of the Effects of Four Freezing Methods on Motility Characteristics, Morphology, and Viability of Postthaw Stallion Epididymal Sperm Stefanie Neuhauser DVM, DECAR a, Svenja Rheinfeld DVM b, Johannes Handler DVM, PhD, DECAR a, * a b
Pferdezentrum Bad Saarow, Equine Reproduction Unit, Freie Universität Berlin, Bad Saarow, Germany Clinic for Horses, Freie Universität Berlin, Berlin, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 December 2013 Received in revised form 22 February 2014 Accepted 4 March 2014 Available online xxxx
Semen cryopreservation is of growing interest in the horse breeding industry, and collecting epididymal sperm might provide the chance to preserve genetic material from valuable stallions after severe injury or death. In case of an unexpected emergency, there may not always be an adequate laboratory nearby. Therefore, we compared fast and slow freezing methods using either a programmable freezer or a styrofoam box filled with liquid nitrogen. Epididymides of 10 stallions were collected immediately after routine castration under general anesthesia. Epididymal spermatozoa were evaluated before and after the freeze-thaw process for motility, viability, morphological, and kinematic parameters. Neither postthaw motility nor kinematic values differed among the four freezing protocols. Morphological abnormalities after freezing and thawing differed among epididymal segments. However, there were significantly more nonviable spermatozoa after the freezethaw process using the fast freezing process in the styrofoam box filled with liquid nitrogen compared with all other freezing processes. According to the results of this study, freezing in nitrogen vapor should be considered as an alternative to the programmable freezer only in combination with a prolonged cooling period. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Semen Horse Preservation Epididymis CASA Bromophenol blue
1. Introduction Semen cryopreservation has been established for horses since the late 1950s [1] and is of growing interest in horse breeding industry because an increasing number of breed associations allowed registration of foals born after insemination with frozen-thawed semen [2,3]. Many studies investigating different extenders and freezing procedures were carried out with the aim at improving fertility rates with frozen-thawed stallion semen [4]. In contrast to
* Corresponding author at: Prof Johannes Handler, DVM, PhD, DECAR, Pferdezentrum Bad Saarow, Equine Reproduction Unit, Freie Universität Berlin, Silberberg 1, 15526 Bad Saarow, Germany. E-mail address: [email protected] (J. Handler). 0737-0806/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2014.03.002
bull semen, the postthaw semen quality varies considerably among individual stallions, which in fact makes it difficult to find one particular freezing protocol useful and successful for all stallions [5]. Preservation of epididymal sperm is useful in endangered species, and the domestic horse can serve as a model for threatened wild equids [6,7]. Furthermore, in case of unexpected injury, which will end the breeding career, or cause death of valuable sires, collecting epididymal sperm might be the last chance to preserve their genetic material. In these cases, it is very important to use an optimal cryopreservation procedure because only a limited amount of epididymal sperm from a particular stallion will be available. Every step of the preservation process influences sperm parameters. Damage of spermatozoa mostly occurs during
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the freezing and thawing process [8,9]. Resistance to cryopreservation injuries depends to a great extent on the composition of the plasma membrane [10]. During maturation along the epididymis, spermatozoa become fertile and motile, but as a consequence of remodeling of the plasma membrane, they also become more susceptible to cold shock [10]. The contact with seminal plasma (SP) during ejaculation initiates a further maturation process, leading to reduced resistance against cooling and freezing injury [9,11]. Therefore, epididymal spermatozoa may need different cooling and freezing protocols than ejaculated sperm. To preserve spermatozoa, special equipment is necessary, but in case of an unexpected emergency, there may not always be an adequate laboratory nearby. Then, a styrofoam box with liquid nitrogen (LN2) could serve as an easy, cost effective, and valuable way to cryopreserve gametes. In this study, we compared four freezing methods using a programmable freezer and a styrofoam box filled with LN2 and a floating rack. We investigated the differences in motion characteristics, morphology, and viability of frozenthawed sperm from three different segments of the cauda epididymidis. 2. Material and Methods 2.1. Experimental Animals Epididymides of 10 healthy stallions of different breeds (Warmblood, Lusitano, Thoroughbred, Trotter, and American Quarter Horse) were collected immediately after standardized routine castration under general anesthesia. Stallions were between 3- and 14-year-old and without history of previous ejaculation. Castration was performed because owners wanted to stop stallion behavior; therefore, it was not possible to compare data of ejaculated and epididymal sperm. Macroscopic examination of the testes and epididymides did not reveal any abnormalities. A 9year-old healthy Shetland pony stallion with poor sperm quality was used as SP donor. 2.2. Collection of Epididymal Sperm The cauda of both epididymides was dissected into three segments (E9dmost caudal, E8dmiddle, and E7dmost cranial segments; [12]). Spermatozoa were harvested by retrograde flush (E9) or mincing and incubation
for 10 minutes at 37 C (E7 and E8). After recovery and incubation using 10-mL Dulbecco phosphate buffered saline (BioWhittaker; Lonza, Verviers, Belgium), semen was centrifuged (600 g; 10 minutes), and the sperm pellet was extended using a commercial skim-milk extender to a concentration of 800 106 sperm/mL (E9) and 80 106 sperm/mL (E7 and E8) (EquiPro; Minitube, Tiefenbach, Germany). Then samples were diluted using an egg-yolk extender with 5% glycerol (Gent freezing extender; Minitube) to a concentration of 400 106 sperm/mL (E9) and 40 106 sperm/mL (E7 and E8). Final dilution of sperm comprised 2.5% of glycerol. Thereafter, semen was filled into 0.5-mL straws. 2.3. Freezing Protocols Freezing was performed using either a programmable freezer or a styrofoam box filled with LN2 and a floating rack. For both methods, we compared a fast and a slow freezing protocol. A total of four different freezing protocols were investigated (Table 1). Using the programmable freezer, for the fast and slow freezing processes, cooling rates of 1.0 and 0.1 C/min between 20 C and 4 C were performed, respectively. From 4 C to 140 C, a freezing rate of 60 C/min was applied. Then all straws were plunged into LN2. For freezing in nitrogen vapor, straws were placed onto the floating freezing rack 5 cm above LN2 surface for 20 minutes, either immediately after packaging at room temperature (fast) or after an equilibration time of 150 minutes at 4 C (slow). Thereafter, the straws were also plunged into LN2. Four aliquots of each segment of each epididymis were randomly allocated to one of the freezing processes. Therefore, the protocols could be compared among stallions and among the three segments of the cauda epididymidis. After at least 1 month of storage in LN2, one straw per epididymal segment per stallion per freezing protocol was thawed at 38 C for 20 seconds. Homologous SP was added to the thawed semen sample resulting in an 80% proportion of SP in the final volume. Then, motion characteristics were analyzed on a prewarmed microscope slide at 37 C. Seminal plasma was collected from a Shetland pony stallion, centrifuged, filtered (Millex GP Filter Unit; Millipore Express PES Membrane 0.22 mm; Millipore, Carrigtwohill, Ireland), stored at 20 C, thawed at room temperature, and prewarmed to 38 C before addition to the semen sample.
Table 1 Freezing protocols Protocol 1
Protocol 2
Protocol 3
Protocol 4
Programmable freezer Fast freezing process 20 C (-1.0 C/min) 4 C 4 C (-60 C/min) -140 LN2 for at least 1 month Thawing: 38 C for 20 sec. Addition of homologous seminal plasma (80%)
Programmable freezer Slow freezing process 20 C (-0.1 C/min) 4 C 4 C (-60 C/min) -140 LN2 for at least 1 month Thawing: 38 C for 20 sec. Addition of homologous seminal plasma (80%)
Nitrogen vapour Fast freezing process 5 cm above LN2 for 20 min LN2 for at least 1 month Thawing: 38 C for 20 sec. Addition of homologous seminal plasma (80%)
Nitrogen vapour Slow freezing process Refrigerator at 4 C for 150 min 5 cm above LN2 for 20 min LN2 for at least 1 month Thawing: 38 C for 20 sec. Addition of homologous seminal plasma (80%)
LN2 means liquid nitrogen; values in brackets are cooling rates.
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2.4. Motility Analysis and Kinematic Characteristics All motility and kinematic parameters were analyzed by a CASA System (Sperm Vision; Minitube) with a 0.63 video-adapter and following settings: 60 frames per second, evaluation of six fields or 3,000 cells, and spermatozoa were detected between 14 and 80 mm2, local motility was considered if distance straight line <6 mm, and linear motion characterized if straightness >90% and linearity >50%. A 6.5-mL drop was placed on a prewarmed microscope slide covered with an 18 18 mm coverslip [13]. Percentage of total and progressive motile sperm, curvilinear velocity (VCL, mm/s), straight-line velocity (VSL, mm/s), average path velocity (VAP, mm/s), amplitude of lateral head displacement (mm), and beat cross frequency (Hz) were evaluated. Motion characteristics were compared between sperm immediately before the freezing process (after centrifugation and the addition of freezing extender to a final glycerol concentration of 2.5%) and frozen-thawed sperm after the addition of SP. 2.5. Sperm Morphology and Viability Analysis Sperm were stained using bromophenol blue and nigrosin, and 100 sperm in each of three different areas on the microscope slide were analyzed. Thus, 300 sperm per sample were classified according to their morphology and viability. Sperm with blue heads were considered membrane damaged and nonviable. Defect sperm heads and tails (principal piece and midpiece), coiled tails, looped tails, isolated heads, and the presence of cytoplasmic droplets were recorded and data of sperm immediately after harvesting (before centrifugation process) were compared with data after the freeze-thaw process. 2.6. Statistics We used JMP statistic software (SAS Institute Inc, Cary, NC) for data analysis. Because data were not normally distributed, nonparametric tests were used. Motility and kinetic values of different freezing protocols, differences among the three segments of the cauda epididymidis, and viability were compared using Wilcoxon signed-rank test. The relation between epididymal segments and freezing procedures were calculated by chi-square test. Viability of
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spermatozoa was compared by the analysis of variances. P values <.05 were considered significant. Data of motion characteristics are given as median and range according to the Andrology Special Interest group of the European Society of Human Reproduction and Embryology [14]. Morphological abnormalities and viability results are given in percentage. 3. Results 3.1. Motility and Kinematic Values Total and progressive motility and kinematic values of spermatozoa of the three different segments of the cauda epididymis before freezing and after the addition of SP to frozen-thawed sperm are presented in Tables 2 and 3. Although there was a marked decrease in total and progressive motility after all freezing processes (P < .05), kinematic parameters did not change (P > .05). Neither postthaw motility (P > .05) nor kinematic values (P > .05) differed among the four freezing protocols. Comparison of epididymal segments yielded no differences of motility among freezing protocols (P < .05), but postthaw velocity values (VCL, VSL, and VAP) differed among different segments in protocols 1 and 2 (P < .05). 3.2. Morphology All values of morphological abnormalities are presented in Table 4. In all segments of the cauda epididymidis, the percentage of abnormal sperm heads was not changed by any freezing protocol. The freeze-thaw processes caused an increase in abnormalities of the principal and midpieces and in the percentage of looped tails, but the percentage of cytoplasmic droplets decreased. Immediately after sperm collection, coiled and looped tails were detected more frequently in the caudal epididymal segment (E9) compared with more cranial segments (E7 and E8). Isolated heads appeared more often in the cranial segments (E7 and E8) of the cauda epididymis than in E9 immediately after sperm collection and after thawing using the slow freezing protocols (protocol 2 and protocol 4; P < .05). In E7 and E8, there were more isolated heads after sperm harvesting than after the freeze-thawing procedure (P < .05). In contrast to E7 and E8, in E9, the incidence of coiled tails and isolated heads did not change after any freeze-thawing process.
Table 2 Total motility (TM) and progressive motility (PM) in percentage before freezing and thawing and after postthaw addition of seminal plasma of three different segments of the cauda epididymidis (E7dcranial, E8dmiddle, and E9dcaudal) Motility
Epididymal Segment
Before Freeze-Thawing
TM
E7 E8 E9 E7 E8 E9
44.34 54.65 46.17 36.84 49.32 38.04
PM
After Freeze-Thawing and Addition of Seminal Plasma Protocol 1
(2.67–89.66)a (6.04–86.36)a (5.82–85.82)a (0.43–83.34)a (2.26–80.14)a (0.77–82.84)a
25.77 22.28 35.07 20.34 17.48 23.53
(4.22–56.35)b (2.49–56.00)b (1.26–74.20)b (2.48–50.46)b (1.92–43.48)b (0.24–67.46)b
Protocol 2 22.36 19.50 19.18 14.26 13.37 12.23
(3.51–46.47)b (1.81–54.28)b (0.99–62.27)b (0.99–37.10)b (0.19–48.86)b (0–55.71)b
Protocol 3 17.46 13.19 10.76 12.69 10.50 6.40
(3.40–34.25)b (2.64–38.42)b (1.79–49.18)b (1.31–30.73)b (1.27–31.18)b (0.16–39.87)b
Data are given as median (range). Superscript lowercase letters (a and b) within rows indicate significant differences (P < .05).
Protocol 4 17.94 14.68 18.23 12.30 10.28 12.43
(2.09–36.33)b (1.37–44.85)b (0.78–43.72)b (0.72–32.14)b (0.64–39.55)b (0.12–34.94)b
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Table 3 Kinematic values before freezing and thawing and after postthaw addition of seminal plasma of three different segments of the cauda epididymidis (E7dcranial, E8dmiddle, and E9dcaudal) Kinematic Value
Epididymal Segment
Before Freeze-Thawing
VCL (mm/s)
E7 E8 E9 E7 E8 E9 E7 E8 E9 E7 E8 E9 E7 E8 E9
155.4 151.6 161.6 61.2 59.3 51.0 84.0 81.8 82.7 3.7 3.8 3.9 35.0 33.9 31.0
VSL (mm/s)
VAP (mm/s)
ALH (mm)
BCF (Hz)
After Freeze-Thawing and Addition of SP Protocol 1
(80.1–206.3) (105.4–188.0) (63.1–198.1) (20.3–101.2) (44.4–77.8) (21.1–69.2) (38.3–118.4) (63.2–100.1) (34.6–96.7) (1.6–5.0) (2.8–4.7) (1.7–5.1) (12.7–43.9) (30.6–38.7) (18.6–34.5)
152.5 147.7 119.4 52.1 53.1 44.9 75.7 75.3 62.0 3.5 3.4 3.2 34.1 35.6 33.2
Protocol 2
(117.9–182.9)A (119.2–162.5)A (69.8–159.6)A (44.0–71.9)A (35.4–64.4)A (27.7–63.9)A (61.7–91.5)A (56.0–87.4)A (36.3–86.8)A (2.8–4.1) (2.7–4.0) (1.6–3.8) (29.7–41.1) (30.9–38.9) (13.4–39.3)
153.9 147.2 127.0 58.2 53.3 48.3 78.7 67.0 64.4 3.6 3.5 3.3 34.7 35.8 33.6
(63.7–202.2)A (43.2–168.1)A (0–181.8)A (22.0–72.9)A (14.1–78.1)A (0–67.2)A (37.7–104.5)A (18.2–99.6)A (0–92.3)A (1.2–4.7) (0.9–4.0) (0–4.0) (11.5–44.3) (10.4–43.6) (0–39.6)
Protocol 3
Protocol 4
144.0 135.0 117.7 53.6 51.4 43.5 74.7 70.5 62.1 3.2 3.1 3.1 35.3 35.5 33.7
142.3 143.0 110.0 50.21 51.12 41.80 73.1 73.9 58.9 3.35 3.4 3.0 35.2 34.7 33.7
(69.2–175.9) (77.7–179.2) (25.9–159.5) (21.4–66.5) (31.6–72.5) (12.3–64.7) (39.3–84.9) (40.2–92.5) (16.1–82.5) (1.3–4.2) (1.7–4.2) (1.06–3.77) (13.7–39.9) (26.7–44.6) (7.4–41.4)
(84.8–169.6)A (57.7–163.8)A (53.1–157.4)A (32.41–70.66) (23.16–67.76) (10.42–55.93) (44.9–85.1)A (33.0–86.5)A (22.5–76.7)A (2.01–3.8) (1.4–3.9) (1.3–3.5) (20.3–51.3) (18.8–47.4) (10.6–39.6)
ALH, amplitude of lateral head displacement; BCF, beat cross frequency; SP, seminal plasma; VAP, average path velocity; VCL, curvilinear velocity; VSL, straight-line velocity. Data are given as median (range). Superscript capital letter (A) within columns indicates significant differences between epididymal segments (P < .05). There was no difference between spermatozoa before and after the freeze-thawing process or among freezing procedures (P < .05).
segments after freeze-thaw process in all groups of different freezing procedure (Fig. 1).
3.3. Viability Percentage of membrane-damaged spermatozoa increased after the freezing and thawing process in all epididymal segments (P < .05). There were significantly more nonviable spermatozoa after the freeze-thawing process using protocol 3 compared with all other freezing processes (P < .05; Fig. 1). Comparing raw sperm in three different epididymal segments, there were significantly more nonviable spermatozoa in E7 compared with E9 (P < .05). No differences were detected among epididymal
4. Discussion In the present study, four freezing protocols did not reveal differences concerning motility parameters in epididymal sperm after freezing and/or thawing and postthaw addition of SP. Hence, cooling rates of 0.1 C/min between 20 C and 4 C had no beneficial effect on motion characteristics after cryopreservation compared
Table 4 Morphology immediately after sperm collection and after freezing and thawing of three different segments of the cauda epididymidis (E7dcranial, E8dmiddle, and E9dcaudal) Morphological Abnormalities (%)
Epididymal Segment
After Sperm Collection
Sperm heads
E7 E8 E9 E7 E8 E9 E7 E8 E9 E7 E8 E9 E7 E8 E9 E7 E8 E9 E7 E8 E9
1.45 (0–3) 1.3 (0.3–4) 1.3 (0.6–4.3) 3.15 (0.6–12.3)a 3.65 (0–10.6)a 2.8 (0.3–7.3)a 13.45 (5.3–80)a 12.8 (1.6–77.6)a 13.45 (3.3–82.3)a 0 (0–3)a,A 0 (0–0.6)a,A 0.15 (0–4.6)a,A 0 (0–2)a,A 0 (0–1.3)a,A 0.6 (0–4.3)A 2.55 (1–7)a,A 1.8 (0–4.6)a,A 0.8 (0–4.3)A 47.1 (4.3–65.6)a 52.1 (8.3–67)a 45.3 (6–62.3)a
Midpiece
Bent tails
Looped tails
Coiled tails
Isolated heads
Cytoplasmic droplet
After Freeze-Thawing Process Protocol 1
Protocol 2
Protocol 3
Protocol 4
1.3 1.3 1.15 7.6 7.15 6.15 13.6 14.8 12.8 1 3.8 4.1 0.3 0.6 0.6 1.15 1 1.15 28.1 33.65 35.15
1.15 1.3 1.3 7.15 8.15 6.6 16.3 15.95 11.6 4.3 4.3 7.8 0.3 0.3 0.6 1.45 1 0.6 30.45 34.5 36.6
1.45 1.45 1 6.6 7.65 5.95 16.15 14.1 12.8 3.15 3.5 6.3 0.3 0.45 0.6 2 1.8 1.3 29.8 31.6 39.3
0.8 1.3 1.3 6.95 7.3 6.15 14.65 17.6 12.8 3.5 5.65 8.8 0.3 0.3 0.6 1.45 1.8 0.6 33.15 34.45 35.15
(0.3–11.3) (0.6–12.3) (0.3–9.3) (2.3–13.6)b (2.6–24.6)b (1.3–20.6)b (3–82)b (1.6–87.3)b (3–84.3)b (0–18.3)b (0–14.3)b (0.3–20)b (0–2)b (0–2.6)b (0–4.6) (0.3–4.3)b (0–3)b (0.3–4) (1.6–60.3)b (0.6–54)b (1.3–59.6)b
(0–4.3) (0–3.6) (0.3–5.3) (2–18)b (0.6–17)b (1–23.6)b (2.3–84)b (2.6–83.3)b (3.3–87.6)b (0–20.3)b (0–18)b (0–22.6)b (0–2.3)b (0–3.3)b (0–3) (0–3.6)b,A (0–3.6)b,A (0–2.6)A (1.3–56.3)b (1–55)b (0.6–51)b
(0–3.3) (0–5.6) (0.3–5.3) (3.3–17)b (2.6–20)b (1–22.3)b (1.6–85.3)b (2.6–86)b (2–86.6)b (0–25)b (0–19)b (0–20)b (0–1.3)b (0–3)b (0–2.6) (0–5)b (0–4.3)b (0–3.3) (1.6–51.6)b (0.3–48.6)b (0.3–58.3)b
(0–6) (0–4.3) (0–4) (1.3–15.3)b (1.3–20.3)b (1.6–17.3)b (1–82.6)b (2–85)b (1–84.6)b (0–23.6)b (0–18.6)b (0–23.6)b (0–2.6)b (0–2)b (0–4.3) (0–4)b,A (0.3–4.3)b,A (0–3)A (1.6–53.6)b (1.6–49)b (1–53.6)b
Data are given as median (range). Different superscript lowercase letters (a and b) within rows indicate significant differences between spermatozoa before and after freeze-thawing process and differences among freezing procedures (P < .05). Superscript capital letter (A) within columns indicates significant differences between epididymal segments (P < .05).
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Fig. 1. Sperm viability (%, membrane-damaged spermatozoa) immediately after harvesting (PBS) and freezing and/or thawing (four different freezing protocols: P1dprogrammable freezer fast; P2dprogrammable freezer slow; P3dnitrogen vapor fast; and P4dnitrogen vapor slow) of sperm obtained from three different segments of the cauda epididymidis (E7dcranial, E8dmiddle, and E9dcaudal). Lowercase letters (a and b) indicate significant difference within epididymal segments (P < .05); asterisks (*) indicate significant difference among epididymal segments (P < .05). PBS, phosphate buffered saline.
with 1.0 C/min in all epididymal segments. This is in accordance to the study of Braun et al [15] who were not able to detect any differences in postthaw motility after different cooling procedures in epididymal or ejaculated spermatozoa. To prevent irreversible damage in equine sperm, slower cooling rates between 20 C and 8 C before starting the freezing process are usually recommended ( 0.05 [16]; 0.1 [17]; 0.4 to 0.2 [18]; equal to 0.3 or less [19]; 0.03 to 0.3 [20]; 0.5 [21]; and 0.3 to 0.2 [22]). In contrast to our findings, a cooling rate of 0.05 C/ min improved total and progressive motility and VCL during cooled storage during different extender treatments compared with 0.5 C/min to 5 C for ejaculated spermatozoa [23]. However, although statistically not significant, protocol 1 applying a cooling rate of 1.0 C/min from 20 C to 4 C yielded the highest motility values of frozen-thawed semen in individual samples. Poor motility after all freezing procedures could be because of inadequate cryoprotective mechanisms of the semen extender for epididymal sperm. An optimal formulation of ingredients of an extender for ejaculated sperm need not necessarily be favorable for epididymal sperm, which is why it might be necessary to adjust the composition of freezing extenders and cooling rates to the requirements of epididymal sperm before the freezing procedure [4]. Differing postthaw velocity values between protocols 1 and 2 of sperm from individual epididymal segments could be the consequence of different cryoprotectant–plasma membrane interactions according to different equilibration time, cooling rates, and maturational stages of sperm. Divergent membrane permeability between sperm cells from epididymal segments and ejaculated sperm also could be responsible for low postthaw motility of epididymal sperm. However, Monteiro et al [24] could find a difference in plasma membrane integrity but not in postthaw motility between epididymal and ejaculated sperm. In a recent study, we analyzed the motility of epididymal spermatozoa after every single step during the
preservation process [25]. Low motility rate after the freeze-thaw process was significantly improved after addition of SP. Therefore, we compared postthaw motility parameters of the four freezing protocols in the present study after addition of SP. Postthaw addition of SP is supposed to increase the ability of epididymal spermatozoa to undergo capacitation [26]. In the present study, only one stallion served as a donor for SP because we focused on the effects of freezing procedures. Because it is well known that SP from different stallions influences the quality of frozenthawed ejaculated spermatozoa [27], its impact on epididymal sperm should be investigated. Although the present study did not yield differences in motility and kinematic values of frozen-thawed sperm, we observed distinctly more nonviable sperm in the fast freezing process in nitrogen vapor compared with the other freezing protocols. Amann and Picket [28] showed that too slow cooling rates cause dehydration of sperm cells whereas too rapid cooling results in intracellular ice formation. In addition, they postulated that epididymal sperm may require different cooling and thawing rates than ejaculated sperm because of different composition and stability of membrane structure [28]. However, the huge variety of freezing methods, extenders, centrifugation time and force, cooling rates, and thawing rates make it difficult to compare studies on cryopreservation of sperm because every single step of sperm processing has the potential to influence sperm quality and postthaw fertility [9,29]. In addition, there is also a tremendous variability in sperm quality and sensitivity to cryopreservation among sperms from individual stallions. Dias Maziero et al [30] compared two freezing processes in ejaculated sperm using either LN2 in a styrofoam box or a programmable freezer. They could not find differences in motility patterns comparing these two methods. In contrast to our study, they could not detect different plasma membrane integrity among the freezing methods. However, in that study, both of the two cooling and freezing protocols, the freezing extender, and the thawing
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process differed from our protocols. Devireddy et al [19] calculated that the optimal cooling rate from 4 C to 80 C is 60 C/min (range, 20 to 100 C/min) for stallion sperm using cryoprotective agents. The temperature within 2.5–5 cm above the LN2 level is 160 C [17] and a freezing rate of 60 C/min was achieved 4 cm above LN2 [31]. Therefore, freezing rates using the programmable freezer and the floating rack on LN2 are comparable, and the temperatures are within the range recommended by Devireddy et al [19]. According to the results of the present study, neither total nor progressive motility varied among epididymal segments after freeze-thawing process, but there were differences in kinematic values. In protocols 1 and 2, velocity values were the lowest in segment E9. This suggested that sperm of the most caudal segment were more susceptible to environmental changes, which might also indicate increased permeability of sperm for bromophenol blue immediately after harvesting. However, there was no difference in morphology or viability among frozenthawed spermatozoa of different segments. After sperm collection, a higher proportion of membrane-damaged spermatozoa were obtained from the more cranial segments of the cauda epididymidis. This may be caused by differing membrane permeability of spermatozoa of more cranial parts of the cauda epididymidis, which disappeared after the freezing and thawing processes. Similarly, isolated heads appeared more frequent in the more cranial segments (E7 and E8) than in E9, which might be a consequence of the different collection methods. In segments E7 and E8, spermatozoa were harvested by mincing the epididymal tissue, whereas spermatozoa of E9 were collected by retrograde flush. Cary et al [32] could not find any difference in morphology, motility, or number of spermatozoa between sperm collection by retrograde flush or flotation technique, in which epididymal tissue was slashed by a scalpel blade. However, maybe the mincing technique using three razor blades was an even more traumatic collection method. In the more cranial epididymal segments (E7 and E8), more isolated heads were observed after sperm harvesting than after the freeze-thaw process. This finding suggests that isolated heads could have been lost by centrifugation after sperm collection. In all segments, more cytoplasmic droplets were detected after sperm harvesting than after freeze-thaw processes. The cytoplasmic droplet is a remnant of germ cell cytoplasm and plays a role in volume regulation and osmolyte transport. Osmotic pressure of the diluent can influence the appearance of the droplet [33]. In a previous study, decrease in percentage of distal cytoplasmic droplets in epididymal sperm after the freeze-thaw process was been reported [34]. The reduction of cytoplasmic droplets indicates that the freeze-thaw process supports the separation of droplets, which may be caused by centrifugation, osmotic stress, or changes in membrane structures similar to “cryocapacitation” [32,35]. In this study, we investigated different cryopreservation processes on epididymal sperm in stallions independent of breed, age, or previous breeding data. In case of unexpected injury of a valuable stallion, it might be important to use a freezing process useful for the majority of stallions.
However, sperm parameters differed tremendously between individual stallions similar to ejaculated stallion sperm. Thus, different preservation procedures depending on individual stallion sperm quality may be beneficial. 5. Conclusion In the present study, we compared a programmable freezer with a floating rack on LN2 surface to cryopreserve stallion epididymal sperm. There was no difference in postthaw motion characteristics of sperm among epididymal segments performing different cooling and freezing regimens, but there was an impact on viability. According to the results of this study, using semen extenders EquiPro and Gent freezing extender, freezing in nitrogen vapor should be considered as an alternative to the programmable freezer only in combination with a prolonged cooling period. Acknowledgments All authors read and approved the manuscript and agree to the submission of the manuscript to the journal. The authors declare no conflict of interest and that no financial and personal relationships with other people or organizations have inappropriately influenced their work. The authors S.N., S.R., and J.H. contributed 60%, 30%, and 10%, respectively, for experimental design; the authors S.N. and S.R. equally contributed for data acquisition; the authors S.N., S.R., and J.H. contributed 20%, 10%, and 70%, respectively, for statistics; the authors S.N. and J.H. equally contributed for data interpretation; and the authors S.N., S.R., and J.H. contributed 70%, 10%, and 20%, respectively, for writing manuscript. References [1] Barker CAV, Gandier JCC. Pregnancy in a mare resulting from frozen epididymal spermatozoa. Can J Comp Med 1957;21:47–51. [2] Loomis PR. The equine frozen semen industry. Anim Reprod Sci 2001;68:191–200. [3] Allen WR. The development and application of the modern reproductive technologies to horse breeding. Reprod Dom Anim 2005;40: 310–29. [4] Sieme H, Harrison RAP, Petrunkina AM. Cryobiological determinants of frozen semen quality, with special reference to stallion. Anim Reprod Sci 2008;107:276–92. [5] Loomis PR, Graham JK. Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols. Anim Reprod Sci 2008;105:119–28. [6] Graham JK, Card C. Preservation of genetics from dead or dying stallions. In: Samper JC, Pycock JF, McKinnon AO, editors. Current therapy in equine reproduction. St. Louis: Saunders Elsevier; 2007. p. 281–4. [7] Smits K, Hoogewijs M, Woelders H, Daels P, Van Soom A. Breeding or assisted reproduction? Relevance of the horse model applied to the conservation of endangered equids. Reprod Dom Anim 2012; 47(Suppl 4):239–48. [8] Blach EL, Amann RP, Bowen RA, Frantz D. Changes in quality of stallion spermatozoa during cryopreservation: plasma membrane integrity and motion characteristics. Theriogenology 1989;31:283–98. [9] Watson PF. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod Fert Dev 1995;7:871–91. [10] White IG. Lipids and calcium uptake of sperm in relation to cold shock and preservation: a review. Reprod Fertil Dev 1993;5:639–58. [11] Töpfer-Petersen E, Ekhlasi-Hundrieser M, Kirchhoff C, Leeb T, Sieme H. The role of stallion seminal proteins in fertilisation. Anim Reprod Sci 2005;89:159–70.
S. Neuhauser et al. / Journal of Equine Veterinary Science xx (2014) 1–7 [12] Fouchécourt S, Dacheux F, Dacheux J-L. Glutathione-independent prostaglandin D2 synthase in ram and stallion epididymal fluids: origin and regulation. Biol Reprod 1999;60:558–66. [13] World Health Organization. Standard procedure. In: Cooper TG, editor. WHO laboratory manual for the examination and processing of human semen. 5th ed. Geneva: WHO Press; 2010. p. 7–114. [14] ESHRE Andrology Special Interest Group. Guidelines on the application of CASA technology in the analysis of spermatozoa. Hum Reprod 1998;13:142–5. [15] Braun J, Sakai M, Hochi S, Oguri N. Preservation of ejaculated and epididymal stallion spermatozoa by cooling and freezing. Theriogenology 1994;41:809–18. [16] Moran DM, Jasko DJ, Squires EL, Amann RP. Determination of temperature and cooling rate which induce cold shock in stallion spermatozoa. Theriogenology 1992;38:999–1012. [17] Heitland AV, Jasko DJ, Squires EL, Graham JK, Pickett BW, Hamilton C. Factors affecting motion characteristics of frozenthawed stallion spermatozoa. Equine Vet J 1996;28:47–53. [18] Vidament M, Ecot P, Noue P, Bourgeois C, Magistrini M, Palmer E. Centrifugation and addition of glycerol at 22 C instead of 4 C improve post-thaw motility and fertility of stallion spermatozoa. Theriogenology 2000;54:907–19. [19] Devireddy RV, Swanlund DJ, Olin T, Vincente W, Troedsson MHT, Bischof JC, et al. Cryopreservation of equine sperm: optimal cooling rates in the presence and absence of cryoprotective agents determined using differential scanning calorimetry. Biol Reprod 2002;66: 222–31. [20] Bader H, Sieme H. Künstliche Besamung beim Pferd. In: Busch W, Waberski D, editors. Künstliche Besamung bei Haus- und Nutztieren. Stuttgart: Schattauer; 2007. p. 224–61. [21] Sanchez R, Gomez I, Samper JC. Artificial insemination with frozen semen. In: Samper JC, editor. Equine breeding management and artificial insemination. 2nd ed. St. Louis: Sauders Elsevier; 2009. p. 175–83. [22] Salazar Jr JL, Teague SR, Love CC, Brinsko SP, Blanchard TL, Varner DD. Effect of cryopreservation protocol on postthaw characteristics of stallion sperm. Theriogenology 2011;76:409–18. [23] Bedford SJ, Graham JK, Amann RP, Squires EL, Pickett BW. Use of two freezing extenders to cool stallion spermatozoa to 5 C with and without seminal plasma. Theriogenology 1995;43:939–53.
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[24] Monteiro GA, Papa FO, Zahn FS, Dellaqua Jr JA, Melo CM, Maziero RRD, et al. Cryopreservation and fertility of ejaculated and epididymal stallion sperm. Anim Reprod Sci 2011;127:197–201. [25] Neuhauser S, Rheinfeld S, Handler J. Motility of fresh and frozenthawed stallion sperm from three segments of the epididymal cauda and the effect of post-thaw seminal plasma addition on motility. J Equine Vet Sci 2013;33:942–9. [26] Sostaric E, Kraan H, Stout TAE. Role of seminal plasma in the attainment of fertilizing capacity by stallion epididymal sperm. Anim Reprod Sci 2010;121S:S184–5. [27] Aurich JE, Kühne A, Hoppe H, Aurich C. Seminal plasma affects membrane integrity and motility of equine spermatozoa after cryopreservation. Theriogenology 1996;46:791–7. [28] Amann RP, Pickett BW. Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. J Equine Vet Sci 1987;7: 145–73. [29] Neild DM, Gadella BM, Chaves MG, Miragaya MH, Colenbrander B, Agüero A. Membrane changes during different stages of a freezethaw protocol for equine semen cryopreservation. Theriogenology 2003;59:1693–705. [30] Dias Maziero RR, Guasti PN, Monteiro GA, Avanzi BR, Hartwig FP, Lisboa FP, et al. Evaluation of sperm kinetics and plasma membrane integrity of frozen equine semen in different storage volumes and freezing conditions. J Equine Vet Sci 2013;33:165–8. [31] Cochran JD, Amann RP, Froman DP, Pickett BW. Effect of centrifugation, glycerol level, cooling to 5 C, freezing rate and thawing rate on the post-thaw motility of equine sperm. Theriogenology 1984; 22:25–38. [32] Cary JA, Madill S, Farnsworth K, Hayna JT, Duoos L, Fahning ML. A comparison of electroejaculation and epididymal sperm collection techniques in stallions. Can Vet J 2004;45:35–41. [33] Cooper TG, Yeung C-H. Acquisition of volume regulatory response of sperm upon maturation in the epididymis and the role of the cytoplasmic droplet. Microsc Res Tech 2003;61:28–38. [34] Heise A, Thompson PN, Gerber D. Influence of seminal plasma on fresh and post-thaw parameters of stallion epididymal sperm. Anim Reprod Sci 2011;123:192–201. [35] Thomas AD, Meyers SA, Ball BA. Capacitation-like changes in equine spermatozoa following cryopreservation. Theriogenology 2006;65: 1531–50.
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Original Research
Comparison of the Effects of Four Freezing Methods on Motility Characteristics, Morphology, and Viability of Postthaw Stallion Epididymal Sperm Stefanie Neuhauser DVM, DECAR a, Svenja Rheinfeld DVM b, Johannes Handler DVM, PhD, DECAR a, * a b
Pferdezentrum Bad Saarow, Equine Reproduction Unit, Freie Universität Berlin, Bad Saarow, Germany Clinic for Horses, Freie Universität Berlin, Berlin, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 December 2013 Received in revised form 22 February 2014 Accepted 4 March 2014 Available online xxxx
Semen cryopreservation is of growing interest in the horse breeding industry, and collecting epididymal sperm might provide the chance to preserve genetic material from valuable stallions after severe injury or death. In case of an unexpected emergency, there may not always be an adequate laboratory nearby. Therefore, we compared fast and slow freezing methods using either a programmable freezer or a styrofoam box filled with liquid nitrogen. Epididymides of 10 stallions were collected immediately after routine castration under general anesthesia. Epididymal spermatozoa were evaluated before and after the freeze-thaw process for motility, viability, morphological, and kinematic parameters. Neither postthaw motility nor kinematic values differed among the four freezing protocols. Morphological abnormalities after freezing and thawing differed among epididymal segments. However, there were significantly more nonviable spermatozoa after the freezethaw process using the fast freezing process in the styrofoam box filled with liquid nitrogen compared with all other freezing processes. According to the results of this study, freezing in nitrogen vapor should be considered as an alternative to the programmable freezer only in combination with a prolonged cooling period. Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Semen Horse Preservation Epididymis CASA Bromophenol blue
1. Introduction Semen cryopreservation has been established for horses since the late 1950s [1] and is of growing interest in horse breeding industry because an increasing number of breed associations allowed registration of foals born after insemination with frozen-thawed semen [2,3]. Many studies investigating different extenders and freezing procedures were carried out with the aim at improving fertility rates with frozen-thawed stallion semen [4]. In contrast to
* Corresponding author at: Prof Johannes Handler, DVM, PhD, DECAR, Pferdezentrum Bad Saarow, Equine Reproduction Unit, Freie Universität Berlin, Silberberg 1, 15526 Bad Saarow, Germany. E-mail address: [email protected] (J. Handler). 0737-0806/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jevs.2014.03.002
bull semen, the postthaw semen quality varies considerably among individual stallions, which in fact makes it difficult to find one particular freezing protocol useful and successful for all stallions [5]. Preservation of epididymal sperm is useful in endangered species, and the domestic horse can serve as a model for threatened wild equids [6,7]. Furthermore, in case of unexpected injury, which will end the breeding career, or cause death of valuable sires, collecting epididymal sperm might be the last chance to preserve their genetic material. In these cases, it is very important to use an optimal cryopreservation procedure because only a limited amount of epididymal sperm from a particular stallion will be available. Every step of the preservation process influences sperm parameters. Damage of spermatozoa mostly occurs during
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the freezing and thawing process [8,9]. Resistance to cryopreservation injuries depends to a great extent on the composition of the plasma membrane [10]. During maturation along the epididymis, spermatozoa become fertile and motile, but as a consequence of remodeling of the plasma membrane, they also become more susceptible to cold shock [10]. The contact with seminal plasma (SP) during ejaculation initiates a further maturation process, leading to reduced resistance against cooling and freezing injury [9,11]. Therefore, epididymal spermatozoa may need different cooling and freezing protocols than ejaculated sperm. To preserve spermatozoa, special equipment is necessary, but in case of an unexpected emergency, there may not always be an adequate laboratory nearby. Then, a styrofoam box with liquid nitrogen (LN2) could serve as an easy, cost effective, and valuable way to cryopreserve gametes. In this study, we compared four freezing methods using a programmable freezer and a styrofoam box filled with LN2 and a floating rack. We investigated the differences in motion characteristics, morphology, and viability of frozenthawed sperm from three different segments of the cauda epididymidis. 2. Material and Methods 2.1. Experimental Animals Epididymides of 10 healthy stallions of different breeds (Warmblood, Lusitano, Thoroughbred, Trotter, and American Quarter Horse) were collected immediately after standardized routine castration under general anesthesia. Stallions were between 3- and 14-year-old and without history of previous ejaculation. Castration was performed because owners wanted to stop stallion behavior; therefore, it was not possible to compare data of ejaculated and epididymal sperm. Macroscopic examination of the testes and epididymides did not reveal any abnormalities. A 9year-old healthy Shetland pony stallion with poor sperm quality was used as SP donor. 2.2. Collection of Epididymal Sperm The cauda of both epididymides was dissected into three segments (E9dmost caudal, E8dmiddle, and E7dmost cranial segments; [12]). Spermatozoa were harvested by retrograde flush (E9) or mincing and incubation
for 10 minutes at 37 C (E7 and E8). After recovery and incubation using 10-mL Dulbecco phosphate buffered saline (BioWhittaker; Lonza, Verviers, Belgium), semen was centrifuged (600 g; 10 minutes), and the sperm pellet was extended using a commercial skim-milk extender to a concentration of 800 106 sperm/mL (E9) and 80 106 sperm/mL (E7 and E8) (EquiPro; Minitube, Tiefenbach, Germany). Then samples were diluted using an egg-yolk extender with 5% glycerol (Gent freezing extender; Minitube) to a concentration of 400 106 sperm/mL (E9) and 40 106 sperm/mL (E7 and E8). Final dilution of sperm comprised 2.5% of glycerol. Thereafter, semen was filled into 0.5-mL straws. 2.3. Freezing Protocols Freezing was performed using either a programmable freezer or a styrofoam box filled with LN2 and a floating rack. For both methods, we compared a fast and a slow freezing protocol. A total of four different freezing protocols were investigated (Table 1). Using the programmable freezer, for the fast and slow freezing processes, cooling rates of 1.0 and 0.1 C/min between 20 C and 4 C were performed, respectively. From 4 C to 140 C, a freezing rate of 60 C/min was applied. Then all straws were plunged into LN2. For freezing in nitrogen vapor, straws were placed onto the floating freezing rack 5 cm above LN2 surface for 20 minutes, either immediately after packaging at room temperature (fast) or after an equilibration time of 150 minutes at 4 C (slow). Thereafter, the straws were also plunged into LN2. Four aliquots of each segment of each epididymis were randomly allocated to one of the freezing processes. Therefore, the protocols could be compared among stallions and among the three segments of the cauda epididymidis. After at least 1 month of storage in LN2, one straw per epididymal segment per stallion per freezing protocol was thawed at 38 C for 20 seconds. Homologous SP was added to the thawed semen sample resulting in an 80% proportion of SP in the final volume. Then, motion characteristics were analyzed on a prewarmed microscope slide at 37 C. Seminal plasma was collected from a Shetland pony stallion, centrifuged, filtered (Millex GP Filter Unit; Millipore Express PES Membrane 0.22 mm; Millipore, Carrigtwohill, Ireland), stored at 20 C, thawed at room temperature, and prewarmed to 38 C before addition to the semen sample.
Table 1 Freezing protocols Protocol 1
Protocol 2
Protocol 3
Protocol 4
Programmable freezer Fast freezing process 20 C (-1.0 C/min) 4 C 4 C (-60 C/min) -140 LN2 for at least 1 month Thawing: 38 C for 20 sec. Addition of homologous seminal plasma (80%)
Programmable freezer Slow freezing process 20 C (-0.1 C/min) 4 C 4 C (-60 C/min) -140 LN2 for at least 1 month Thawing: 38 C for 20 sec. Addition of homologous seminal plasma (80%)
Nitrogen vapour Fast freezing process 5 cm above LN2 for 20 min LN2 for at least 1 month Thawing: 38 C for 20 sec. Addition of homologous seminal plasma (80%)
Nitrogen vapour Slow freezing process Refrigerator at 4 C for 150 min 5 cm above LN2 for 20 min LN2 for at least 1 month Thawing: 38 C for 20 sec. Addition of homologous seminal plasma (80%)
LN2 means liquid nitrogen; values in brackets are cooling rates.
S. Neuhauser et al. / Journal of Equine Veterinary Science xx (2014) 1–7
2.4. Motility Analysis and Kinematic Characteristics All motility and kinematic parameters were analyzed by a CASA System (Sperm Vision; Minitube) with a 0.63 video-adapter and following settings: 60 frames per second, evaluation of six fields or 3,000 cells, and spermatozoa were detected between 14 and 80 mm2, local motility was considered if distance straight line <6 mm, and linear motion characterized if straightness >90% and linearity >50%. A 6.5-mL drop was placed on a prewarmed microscope slide covered with an 18 18 mm coverslip [13]. Percentage of total and progressive motile sperm, curvilinear velocity (VCL, mm/s), straight-line velocity (VSL, mm/s), average path velocity (VAP, mm/s), amplitude of lateral head displacement (mm), and beat cross frequency (Hz) were evaluated. Motion characteristics were compared between sperm immediately before the freezing process (after centrifugation and the addition of freezing extender to a final glycerol concentration of 2.5%) and frozen-thawed sperm after the addition of SP. 2.5. Sperm Morphology and Viability Analysis Sperm were stained using bromophenol blue and nigrosin, and 100 sperm in each of three different areas on the microscope slide were analyzed. Thus, 300 sperm per sample were classified according to their morphology and viability. Sperm with blue heads were considered membrane damaged and nonviable. Defect sperm heads and tails (principal piece and midpiece), coiled tails, looped tails, isolated heads, and the presence of cytoplasmic droplets were recorded and data of sperm immediately after harvesting (before centrifugation process) were compared with data after the freeze-thaw process. 2.6. Statistics We used JMP statistic software (SAS Institute Inc, Cary, NC) for data analysis. Because data were not normally distributed, nonparametric tests were used. Motility and kinetic values of different freezing protocols, differences among the three segments of the cauda epididymidis, and viability were compared using Wilcoxon signed-rank test. The relation between epididymal segments and freezing procedures were calculated by chi-square test. Viability of
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spermatozoa was compared by the analysis of variances. P values <.05 were considered significant. Data of motion characteristics are given as median and range according to the Andrology Special Interest group of the European Society of Human Reproduction and Embryology [14]. Morphological abnormalities and viability results are given in percentage. 3. Results 3.1. Motility and Kinematic Values Total and progressive motility and kinematic values of spermatozoa of the three different segments of the cauda epididymis before freezing and after the addition of SP to frozen-thawed sperm are presented in Tables 2 and 3. Although there was a marked decrease in total and progressive motility after all freezing processes (P < .05), kinematic parameters did not change (P > .05). Neither postthaw motility (P > .05) nor kinematic values (P > .05) differed among the four freezing protocols. Comparison of epididymal segments yielded no differences of motility among freezing protocols (P < .05), but postthaw velocity values (VCL, VSL, and VAP) differed among different segments in protocols 1 and 2 (P < .05). 3.2. Morphology All values of morphological abnormalities are presented in Table 4. In all segments of the cauda epididymidis, the percentage of abnormal sperm heads was not changed by any freezing protocol. The freeze-thaw processes caused an increase in abnormalities of the principal and midpieces and in the percentage of looped tails, but the percentage of cytoplasmic droplets decreased. Immediately after sperm collection, coiled and looped tails were detected more frequently in the caudal epididymal segment (E9) compared with more cranial segments (E7 and E8). Isolated heads appeared more often in the cranial segments (E7 and E8) of the cauda epididymis than in E9 immediately after sperm collection and after thawing using the slow freezing protocols (protocol 2 and protocol 4; P < .05). In E7 and E8, there were more isolated heads after sperm harvesting than after the freeze-thawing procedure (P < .05). In contrast to E7 and E8, in E9, the incidence of coiled tails and isolated heads did not change after any freeze-thawing process.
Table 2 Total motility (TM) and progressive motility (PM) in percentage before freezing and thawing and after postthaw addition of seminal plasma of three different segments of the cauda epididymidis (E7dcranial, E8dmiddle, and E9dcaudal) Motility
Epididymal Segment
Before Freeze-Thawing
TM
E7 E8 E9 E7 E8 E9
44.34 54.65 46.17 36.84 49.32 38.04
PM
After Freeze-Thawing and Addition of Seminal Plasma Protocol 1
(2.67–89.66)a (6.04–86.36)a (5.82–85.82)a (0.43–83.34)a (2.26–80.14)a (0.77–82.84)a
25.77 22.28 35.07 20.34 17.48 23.53
(4.22–56.35)b (2.49–56.00)b (1.26–74.20)b (2.48–50.46)b (1.92–43.48)b (0.24–67.46)b
Protocol 2 22.36 19.50 19.18 14.26 13.37 12.23
(3.51–46.47)b (1.81–54.28)b (0.99–62.27)b (0.99–37.10)b (0.19–48.86)b (0–55.71)b
Protocol 3 17.46 13.19 10.76 12.69 10.50 6.40
(3.40–34.25)b (2.64–38.42)b (1.79–49.18)b (1.31–30.73)b (1.27–31.18)b (0.16–39.87)b
Data are given as median (range). Superscript lowercase letters (a and b) within rows indicate significant differences (P < .05).
Protocol 4 17.94 14.68 18.23 12.30 10.28 12.43
(2.09–36.33)b (1.37–44.85)b (0.78–43.72)b (0.72–32.14)b (0.64–39.55)b (0.12–34.94)b
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Table 3 Kinematic values before freezing and thawing and after postthaw addition of seminal plasma of three different segments of the cauda epididymidis (E7dcranial, E8dmiddle, and E9dcaudal) Kinematic Value
Epididymal Segment
Before Freeze-Thawing
VCL (mm/s)
E7 E8 E9 E7 E8 E9 E7 E8 E9 E7 E8 E9 E7 E8 E9
155.4 151.6 161.6 61.2 59.3 51.0 84.0 81.8 82.7 3.7 3.8 3.9 35.0 33.9 31.0
VSL (mm/s)
VAP (mm/s)
ALH (mm)
BCF (Hz)
After Freeze-Thawing and Addition of SP Protocol 1
(80.1–206.3) (105.4–188.0) (63.1–198.1) (20.3–101.2) (44.4–77.8) (21.1–69.2) (38.3–118.4) (63.2–100.1) (34.6–96.7) (1.6–5.0) (2.8–4.7) (1.7–5.1) (12.7–43.9) (30.6–38.7) (18.6–34.5)
152.5 147.7 119.4 52.1 53.1 44.9 75.7 75.3 62.0 3.5 3.4 3.2 34.1 35.6 33.2
Protocol 2
(117.9–182.9)A (119.2–162.5)A (69.8–159.6)A (44.0–71.9)A (35.4–64.4)A (27.7–63.9)A (61.7–91.5)A (56.0–87.4)A (36.3–86.8)A (2.8–4.1) (2.7–4.0) (1.6–3.8) (29.7–41.1) (30.9–38.9) (13.4–39.3)
153.9 147.2 127.0 58.2 53.3 48.3 78.7 67.0 64.4 3.6 3.5 3.3 34.7 35.8 33.6
(63.7–202.2)A (43.2–168.1)A (0–181.8)A (22.0–72.9)A (14.1–78.1)A (0–67.2)A (37.7–104.5)A (18.2–99.6)A (0–92.3)A (1.2–4.7) (0.9–4.0) (0–4.0) (11.5–44.3) (10.4–43.6) (0–39.6)
Protocol 3
Protocol 4
144.0 135.0 117.7 53.6 51.4 43.5 74.7 70.5 62.1 3.2 3.1 3.1 35.3 35.5 33.7
142.3 143.0 110.0 50.21 51.12 41.80 73.1 73.9 58.9 3.35 3.4 3.0 35.2 34.7 33.7
(69.2–175.9) (77.7–179.2) (25.9–159.5) (21.4–66.5) (31.6–72.5) (12.3–64.7) (39.3–84.9) (40.2–92.5) (16.1–82.5) (1.3–4.2) (1.7–4.2) (1.06–3.77) (13.7–39.9) (26.7–44.6) (7.4–41.4)
(84.8–169.6)A (57.7–163.8)A (53.1–157.4)A (32.41–70.66) (23.16–67.76) (10.42–55.93) (44.9–85.1)A (33.0–86.5)A (22.5–76.7)A (2.01–3.8) (1.4–3.9) (1.3–3.5) (20.3–51.3) (18.8–47.4) (10.6–39.6)
ALH, amplitude of lateral head displacement; BCF, beat cross frequency; SP, seminal plasma; VAP, average path velocity; VCL, curvilinear velocity; VSL, straight-line velocity. Data are given as median (range). Superscript capital letter (A) within columns indicates significant differences between epididymal segments (P < .05). There was no difference between spermatozoa before and after the freeze-thawing process or among freezing procedures (P < .05).
segments after freeze-thaw process in all groups of different freezing procedure (Fig. 1).
3.3. Viability Percentage of membrane-damaged spermatozoa increased after the freezing and thawing process in all epididymal segments (P < .05). There were significantly more nonviable spermatozoa after the freeze-thawing process using protocol 3 compared with all other freezing processes (P < .05; Fig. 1). Comparing raw sperm in three different epididymal segments, there were significantly more nonviable spermatozoa in E7 compared with E9 (P < .05). No differences were detected among epididymal
4. Discussion In the present study, four freezing protocols did not reveal differences concerning motility parameters in epididymal sperm after freezing and/or thawing and postthaw addition of SP. Hence, cooling rates of 0.1 C/min between 20 C and 4 C had no beneficial effect on motion characteristics after cryopreservation compared
Table 4 Morphology immediately after sperm collection and after freezing and thawing of three different segments of the cauda epididymidis (E7dcranial, E8dmiddle, and E9dcaudal) Morphological Abnormalities (%)
Epididymal Segment
After Sperm Collection
Sperm heads
E7 E8 E9 E7 E8 E9 E7 E8 E9 E7 E8 E9 E7 E8 E9 E7 E8 E9 E7 E8 E9
1.45 (0–3) 1.3 (0.3–4) 1.3 (0.6–4.3) 3.15 (0.6–12.3)a 3.65 (0–10.6)a 2.8 (0.3–7.3)a 13.45 (5.3–80)a 12.8 (1.6–77.6)a 13.45 (3.3–82.3)a 0 (0–3)a,A 0 (0–0.6)a,A 0.15 (0–4.6)a,A 0 (0–2)a,A 0 (0–1.3)a,A 0.6 (0–4.3)A 2.55 (1–7)a,A 1.8 (0–4.6)a,A 0.8 (0–4.3)A 47.1 (4.3–65.6)a 52.1 (8.3–67)a 45.3 (6–62.3)a
Midpiece
Bent tails
Looped tails
Coiled tails
Isolated heads
Cytoplasmic droplet
After Freeze-Thawing Process Protocol 1
Protocol 2
Protocol 3
Protocol 4
1.3 1.3 1.15 7.6 7.15 6.15 13.6 14.8 12.8 1 3.8 4.1 0.3 0.6 0.6 1.15 1 1.15 28.1 33.65 35.15
1.15 1.3 1.3 7.15 8.15 6.6 16.3 15.95 11.6 4.3 4.3 7.8 0.3 0.3 0.6 1.45 1 0.6 30.45 34.5 36.6
1.45 1.45 1 6.6 7.65 5.95 16.15 14.1 12.8 3.15 3.5 6.3 0.3 0.45 0.6 2 1.8 1.3 29.8 31.6 39.3
0.8 1.3 1.3 6.95 7.3 6.15 14.65 17.6 12.8 3.5 5.65 8.8 0.3 0.3 0.6 1.45 1.8 0.6 33.15 34.45 35.15
(0.3–11.3) (0.6–12.3) (0.3–9.3) (2.3–13.6)b (2.6–24.6)b (1.3–20.6)b (3–82)b (1.6–87.3)b (3–84.3)b (0–18.3)b (0–14.3)b (0.3–20)b (0–2)b (0–2.6)b (0–4.6) (0.3–4.3)b (0–3)b (0.3–4) (1.6–60.3)b (0.6–54)b (1.3–59.6)b
(0–4.3) (0–3.6) (0.3–5.3) (2–18)b (0.6–17)b (1–23.6)b (2.3–84)b (2.6–83.3)b (3.3–87.6)b (0–20.3)b (0–18)b (0–22.6)b (0–2.3)b (0–3.3)b (0–3) (0–3.6)b,A (0–3.6)b,A (0–2.6)A (1.3–56.3)b (1–55)b (0.6–51)b
(0–3.3) (0–5.6) (0.3–5.3) (3.3–17)b (2.6–20)b (1–22.3)b (1.6–85.3)b (2.6–86)b (2–86.6)b (0–25)b (0–19)b (0–20)b (0–1.3)b (0–3)b (0–2.6) (0–5)b (0–4.3)b (0–3.3) (1.6–51.6)b (0.3–48.6)b (0.3–58.3)b
(0–6) (0–4.3) (0–4) (1.3–15.3)b (1.3–20.3)b (1.6–17.3)b (1–82.6)b (2–85)b (1–84.6)b (0–23.6)b (0–18.6)b (0–23.6)b (0–2.6)b (0–2)b (0–4.3) (0–4)b,A (0.3–4.3)b,A (0–3)A (1.6–53.6)b (1.6–49)b (1–53.6)b
Data are given as median (range). Different superscript lowercase letters (a and b) within rows indicate significant differences between spermatozoa before and after freeze-thawing process and differences among freezing procedures (P < .05). Superscript capital letter (A) within columns indicates significant differences between epididymal segments (P < .05).
S. Neuhauser et al. / Journal of Equine Veterinary Science xx (2014) 1–7
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Fig. 1. Sperm viability (%, membrane-damaged spermatozoa) immediately after harvesting (PBS) and freezing and/or thawing (four different freezing protocols: P1dprogrammable freezer fast; P2dprogrammable freezer slow; P3dnitrogen vapor fast; and P4dnitrogen vapor slow) of sperm obtained from three different segments of the cauda epididymidis (E7dcranial, E8dmiddle, and E9dcaudal). Lowercase letters (a and b) indicate significant difference within epididymal segments (P < .05); asterisks (*) indicate significant difference among epididymal segments (P < .05). PBS, phosphate buffered saline.
with 1.0 C/min in all epididymal segments. This is in accordance to the study of Braun et al [15] who were not able to detect any differences in postthaw motility after different cooling procedures in epididymal or ejaculated spermatozoa. To prevent irreversible damage in equine sperm, slower cooling rates between 20 C and 8 C before starting the freezing process are usually recommended ( 0.05 [16]; 0.1 [17]; 0.4 to 0.2 [18]; equal to 0.3 or less [19]; 0.03 to 0.3 [20]; 0.5 [21]; and 0.3 to 0.2 [22]). In contrast to our findings, a cooling rate of 0.05 C/ min improved total and progressive motility and VCL during cooled storage during different extender treatments compared with 0.5 C/min to 5 C for ejaculated spermatozoa [23]. However, although statistically not significant, protocol 1 applying a cooling rate of 1.0 C/min from 20 C to 4 C yielded the highest motility values of frozen-thawed semen in individual samples. Poor motility after all freezing procedures could be because of inadequate cryoprotective mechanisms of the semen extender for epididymal sperm. An optimal formulation of ingredients of an extender for ejaculated sperm need not necessarily be favorable for epididymal sperm, which is why it might be necessary to adjust the composition of freezing extenders and cooling rates to the requirements of epididymal sperm before the freezing procedure [4]. Differing postthaw velocity values between protocols 1 and 2 of sperm from individual epididymal segments could be the consequence of different cryoprotectant–plasma membrane interactions according to different equilibration time, cooling rates, and maturational stages of sperm. Divergent membrane permeability between sperm cells from epididymal segments and ejaculated sperm also could be responsible for low postthaw motility of epididymal sperm. However, Monteiro et al [24] could find a difference in plasma membrane integrity but not in postthaw motility between epididymal and ejaculated sperm. In a recent study, we analyzed the motility of epididymal spermatozoa after every single step during the
preservation process [25]. Low motility rate after the freeze-thaw process was significantly improved after addition of SP. Therefore, we compared postthaw motility parameters of the four freezing protocols in the present study after addition of SP. Postthaw addition of SP is supposed to increase the ability of epididymal spermatozoa to undergo capacitation [26]. In the present study, only one stallion served as a donor for SP because we focused on the effects of freezing procedures. Because it is well known that SP from different stallions influences the quality of frozenthawed ejaculated spermatozoa [27], its impact on epididymal sperm should be investigated. Although the present study did not yield differences in motility and kinematic values of frozen-thawed sperm, we observed distinctly more nonviable sperm in the fast freezing process in nitrogen vapor compared with the other freezing protocols. Amann and Picket [28] showed that too slow cooling rates cause dehydration of sperm cells whereas too rapid cooling results in intracellular ice formation. In addition, they postulated that epididymal sperm may require different cooling and thawing rates than ejaculated sperm because of different composition and stability of membrane structure [28]. However, the huge variety of freezing methods, extenders, centrifugation time and force, cooling rates, and thawing rates make it difficult to compare studies on cryopreservation of sperm because every single step of sperm processing has the potential to influence sperm quality and postthaw fertility [9,29]. In addition, there is also a tremendous variability in sperm quality and sensitivity to cryopreservation among sperms from individual stallions. Dias Maziero et al [30] compared two freezing processes in ejaculated sperm using either LN2 in a styrofoam box or a programmable freezer. They could not find differences in motility patterns comparing these two methods. In contrast to our study, they could not detect different plasma membrane integrity among the freezing methods. However, in that study, both of the two cooling and freezing protocols, the freezing extender, and the thawing
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S. Neuhauser et al. / Journal of Equine Veterinary Science xx (2014) 1–7
process differed from our protocols. Devireddy et al [19] calculated that the optimal cooling rate from 4 C to 80 C is 60 C/min (range, 20 to 100 C/min) for stallion sperm using cryoprotective agents. The temperature within 2.5–5 cm above the LN2 level is 160 C [17] and a freezing rate of 60 C/min was achieved 4 cm above LN2 [31]. Therefore, freezing rates using the programmable freezer and the floating rack on LN2 are comparable, and the temperatures are within the range recommended by Devireddy et al [19]. According to the results of the present study, neither total nor progressive motility varied among epididymal segments after freeze-thawing process, but there were differences in kinematic values. In protocols 1 and 2, velocity values were the lowest in segment E9. This suggested that sperm of the most caudal segment were more susceptible to environmental changes, which might also indicate increased permeability of sperm for bromophenol blue immediately after harvesting. However, there was no difference in morphology or viability among frozenthawed spermatozoa of different segments. After sperm collection, a higher proportion of membrane-damaged spermatozoa were obtained from the more cranial segments of the cauda epididymidis. This may be caused by differing membrane permeability of spermatozoa of more cranial parts of the cauda epididymidis, which disappeared after the freezing and thawing processes. Similarly, isolated heads appeared more frequent in the more cranial segments (E7 and E8) than in E9, which might be a consequence of the different collection methods. In segments E7 and E8, spermatozoa were harvested by mincing the epididymal tissue, whereas spermatozoa of E9 were collected by retrograde flush. Cary et al [32] could not find any difference in morphology, motility, or number of spermatozoa between sperm collection by retrograde flush or flotation technique, in which epididymal tissue was slashed by a scalpel blade. However, maybe the mincing technique using three razor blades was an even more traumatic collection method. In the more cranial epididymal segments (E7 and E8), more isolated heads were observed after sperm harvesting than after the freeze-thaw process. This finding suggests that isolated heads could have been lost by centrifugation after sperm collection. In all segments, more cytoplasmic droplets were detected after sperm harvesting than after freeze-thaw processes. The cytoplasmic droplet is a remnant of germ cell cytoplasm and plays a role in volume regulation and osmolyte transport. Osmotic pressure of the diluent can influence the appearance of the droplet [33]. In a previous study, decrease in percentage of distal cytoplasmic droplets in epididymal sperm after the freeze-thaw process was been reported [34]. The reduction of cytoplasmic droplets indicates that the freeze-thaw process supports the separation of droplets, which may be caused by centrifugation, osmotic stress, or changes in membrane structures similar to “cryocapacitation” [32,35]. In this study, we investigated different cryopreservation processes on epididymal sperm in stallions independent of breed, age, or previous breeding data. In case of unexpected injury of a valuable stallion, it might be important to use a freezing process useful for the majority of stallions.
However, sperm parameters differed tremendously between individual stallions similar to ejaculated stallion sperm. Thus, different preservation procedures depending on individual stallion sperm quality may be beneficial. 5. Conclusion In the present study, we compared a programmable freezer with a floating rack on LN2 surface to cryopreserve stallion epididymal sperm. There was no difference in postthaw motion characteristics of sperm among epididymal segments performing different cooling and freezing regimens, but there was an impact on viability. According to the results of this study, using semen extenders EquiPro and Gent freezing extender, freezing in nitrogen vapor should be considered as an alternative to the programmable freezer only in combination with a prolonged cooling period. Acknowledgments All authors read and approved the manuscript and agree to the submission of the manuscript to the journal. The authors declare no conflict of interest and that no financial and personal relationships with other people or organizations have inappropriately influenced their work. The authors S.N., S.R., and J.H. contributed 60%, 30%, and 10%, respectively, for experimental design; the authors S.N. and S.R. equally contributed for data acquisition; the authors S.N., S.R., and J.H. contributed 20%, 10%, and 70%, respectively, for statistics; the authors S.N. and J.H. equally contributed for data interpretation; and the authors S.N., S.R., and J.H. contributed 70%, 10%, and 20%, respectively, for writing manuscript. References [1] Barker CAV, Gandier JCC. Pregnancy in a mare resulting from frozen epididymal spermatozoa. Can J Comp Med 1957;21:47–51. [2] Loomis PR. The equine frozen semen industry. Anim Reprod Sci 2001;68:191–200. [3] Allen WR. The development and application of the modern reproductive technologies to horse breeding. Reprod Dom Anim 2005;40: 310–29. [4] Sieme H, Harrison RAP, Petrunkina AM. Cryobiological determinants of frozen semen quality, with special reference to stallion. Anim Reprod Sci 2008;107:276–92. [5] Loomis PR, Graham JK. Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols. Anim Reprod Sci 2008;105:119–28. [6] Graham JK, Card C. Preservation of genetics from dead or dying stallions. In: Samper JC, Pycock JF, McKinnon AO, editors. Current therapy in equine reproduction. St. Louis: Saunders Elsevier; 2007. p. 281–4. [7] Smits K, Hoogewijs M, Woelders H, Daels P, Van Soom A. Breeding or assisted reproduction? Relevance of the horse model applied to the conservation of endangered equids. Reprod Dom Anim 2012; 47(Suppl 4):239–48. [8] Blach EL, Amann RP, Bowen RA, Frantz D. Changes in quality of stallion spermatozoa during cryopreservation: plasma membrane integrity and motion characteristics. Theriogenology 1989;31:283–98. [9] Watson PF. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reprod Fert Dev 1995;7:871–91. [10] White IG. Lipids and calcium uptake of sperm in relation to cold shock and preservation: a review. Reprod Fertil Dev 1993;5:639–58. [11] Töpfer-Petersen E, Ekhlasi-Hundrieser M, Kirchhoff C, Leeb T, Sieme H. The role of stallion seminal proteins in fertilisation. Anim Reprod Sci 2005;89:159–70.
S. Neuhauser et al. / Journal of Equine Veterinary Science xx (2014) 1–7 [12] Fouchécourt S, Dacheux F, Dacheux J-L. Glutathione-independent prostaglandin D2 synthase in ram and stallion epididymal fluids: origin and regulation. Biol Reprod 1999;60:558–66. [13] World Health Organization. Standard procedure. In: Cooper TG, editor. WHO laboratory manual for the examination and processing of human semen. 5th ed. Geneva: WHO Press; 2010. p. 7–114. [14] ESHRE Andrology Special Interest Group. Guidelines on the application of CASA technology in the analysis of spermatozoa. Hum Reprod 1998;13:142–5. [15] Braun J, Sakai M, Hochi S, Oguri N. Preservation of ejaculated and epididymal stallion spermatozoa by cooling and freezing. Theriogenology 1994;41:809–18. [16] Moran DM, Jasko DJ, Squires EL, Amann RP. Determination of temperature and cooling rate which induce cold shock in stallion spermatozoa. Theriogenology 1992;38:999–1012. [17] Heitland AV, Jasko DJ, Squires EL, Graham JK, Pickett BW, Hamilton C. Factors affecting motion characteristics of frozenthawed stallion spermatozoa. Equine Vet J 1996;28:47–53. [18] Vidament M, Ecot P, Noue P, Bourgeois C, Magistrini M, Palmer E. Centrifugation and addition of glycerol at 22 C instead of 4 C improve post-thaw motility and fertility of stallion spermatozoa. Theriogenology 2000;54:907–19. [19] Devireddy RV, Swanlund DJ, Olin T, Vincente W, Troedsson MHT, Bischof JC, et al. Cryopreservation of equine sperm: optimal cooling rates in the presence and absence of cryoprotective agents determined using differential scanning calorimetry. Biol Reprod 2002;66: 222–31. [20] Bader H, Sieme H. Künstliche Besamung beim Pferd. In: Busch W, Waberski D, editors. Künstliche Besamung bei Haus- und Nutztieren. Stuttgart: Schattauer; 2007. p. 224–61. [21] Sanchez R, Gomez I, Samper JC. Artificial insemination with frozen semen. In: Samper JC, editor. Equine breeding management and artificial insemination. 2nd ed. St. Louis: Sauders Elsevier; 2009. p. 175–83. [22] Salazar Jr JL, Teague SR, Love CC, Brinsko SP, Blanchard TL, Varner DD. Effect of cryopreservation protocol on postthaw characteristics of stallion sperm. Theriogenology 2011;76:409–18. [23] Bedford SJ, Graham JK, Amann RP, Squires EL, Pickett BW. Use of two freezing extenders to cool stallion spermatozoa to 5 C with and without seminal plasma. Theriogenology 1995;43:939–53.
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[24] Monteiro GA, Papa FO, Zahn FS, Dellaqua Jr JA, Melo CM, Maziero RRD, et al. Cryopreservation and fertility of ejaculated and epididymal stallion sperm. Anim Reprod Sci 2011;127:197–201. [25] Neuhauser S, Rheinfeld S, Handler J. Motility of fresh and frozenthawed stallion sperm from three segments of the epididymal cauda and the effect of post-thaw seminal plasma addition on motility. J Equine Vet Sci 2013;33:942–9. [26] Sostaric E, Kraan H, Stout TAE. Role of seminal plasma in the attainment of fertilizing capacity by stallion epididymal sperm. Anim Reprod Sci 2010;121S:S184–5. [27] Aurich JE, Kühne A, Hoppe H, Aurich C. Seminal plasma affects membrane integrity and motility of equine spermatozoa after cryopreservation. Theriogenology 1996;46:791–7. [28] Amann RP, Pickett BW. Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. J Equine Vet Sci 1987;7: 145–73. [29] Neild DM, Gadella BM, Chaves MG, Miragaya MH, Colenbrander B, Agüero A. Membrane changes during different stages of a freezethaw protocol for equine semen cryopreservation. Theriogenology 2003;59:1693–705. [30] Dias Maziero RR, Guasti PN, Monteiro GA, Avanzi BR, Hartwig FP, Lisboa FP, et al. Evaluation of sperm kinetics and plasma membrane integrity of frozen equine semen in different storage volumes and freezing conditions. J Equine Vet Sci 2013;33:165–8. [31] Cochran JD, Amann RP, Froman DP, Pickett BW. Effect of centrifugation, glycerol level, cooling to 5 C, freezing rate and thawing rate on the post-thaw motility of equine sperm. Theriogenology 1984; 22:25–38. [32] Cary JA, Madill S, Farnsworth K, Hayna JT, Duoos L, Fahning ML. A comparison of electroejaculation and epididymal sperm collection techniques in stallions. Can Vet J 2004;45:35–41. [33] Cooper TG, Yeung C-H. Acquisition of volume regulatory response of sperm upon maturation in the epididymis and the role of the cytoplasmic droplet. Microsc Res Tech 2003;61:28–38. [34] Heise A, Thompson PN, Gerber D. Influence of seminal plasma on fresh and post-thaw parameters of stallion epididymal sperm. Anim Reprod Sci 2011;123:192–201. [35] Thomas AD, Meyers SA, Ball BA. Capacitation-like changes in equine spermatozoa following cryopreservation. Theriogenology 2006;65: 1531–50.