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Impact of holding and equilibration time on post-thaw quality of shipped boar semen
Animal Reproduction Science xxx (xxxx) xxx–xxx
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Impact of holding and equilibration time on post-thaw quality of shipped boar semen ⁎
J. Schäfera,b, , D. Waberskib, M. Junga, M. Schulzea a b
Institute for the Reproduction of Farm Animals Schönow e.V. (IFN), Bernauer Allee 10, D-16321 Bernau, Germany Unit for Reproductive Medicine of Clinics/Clinic for Pigs and Small Ruminants, University of Veterinary Medicine Hannover, Hannover, Germany
AR TI CLE I NF O
AB S T R A CT
Keywords: Boar semen Cryopreservation Equilibration Holding time Shipping
Cryopreservation of boar semen is of growing interest for breeding companies. Overnight-shipping of pre-diluted ejaculates to specialized laboratories offers a practicable method, but requires fine-tuned protocols. In this study, the impact of holding post shipping at 17 °C for 2 or 24 h (n = 10 samples) and of equilibration in lactose-egg yolk extender without glycerol at 5 °C for 2, 4, 24 or 48 h (n = 11 samples) before freezing was investigated. Sperm-rich fractions of ejaculates from 21 mature Pietrain boars were collected at a single boar stud. After pre-dilution (1 + 1, v:v) with Beltsville thawing solution, samples were sent to the laboratory. Temperature profiles during transport and initial equilibration time were recorded. Semen quality post-thaw (PT) was evaluated using CASA and flow cytometry. Holding of 2 h after shipping resulted in higher sperm motility (P = 0.013) and beat cross frequency (BCF; P = 0.047) compared to 24 h. Differences between both groups vanished with prolonged incubation at 38 °C PT. Equilibration at 5 °C for 4 h yielded the highest motility and BCF, whereas the equilibration for 48 h impaired sperm motility. Membrane integrity, mitochondrial activity and DNA fragmentation index were not affected by any protocol modification. In conclusion, processing of pre-diluted boar semen shipped overnight within 2 h after arrival at the laboratory is preferred to 24 h of additional holding at 17 °C. Extending the equilibration period in lactose-egg yolk extender without glycerol at 5 °C from 2 h to 4 h before freezing is recommended.
1. Introduction Demand on freezing valuable boars’ ejaculates for international trade and gene banking is high (Men et al., 2012; Knox, 2016). Worldwide less than 1% artificial inseminations (AI) in pigs are performed with frozen-thawed semen (Rodríguez-Gil and Estrada, 2013). Due to the composition of sperm membranes with high protein to phospholipid and low cholesterol to phospholipid ratios, boar spermatozoa are highly sensitive to low temperatures. Chilling injury is expressed as loss of motility, membrane integrity, mitochondrial activity and other sperm functions (Parks and Lynch, 1992; Maxwell and Johnson, 1997). Cryopreservation protocols for boar semen are delicate and complex in order to minimize sperm damage during processing and consequently are often not practicable in the daily routine of AI stations. Shipping of pre-diluted ejaculates to a central personally and technically well-equipped laboratory would allow freezing under ideal conditions thus offering a feasible, cost-efficient method for breeding companies. Overnight shipping inevitably introduces a holding time of extended semen for approximately 24 h at 17 °C before further processing. The effects of holding time at 15–17 °C prior to cryopreservation have been discussed with different outcomes. It is assumed, that during holding time an alteration of the plasma membrane lipid composition and protein phosphorylation takes place, rendering the ⁎
Corresponding author at: Institute for the Reproduction of Farm Animals Schönow e.V. (IFN), Bernauer Allee 10, D-16321 Bernau, Germany. E-mail address: [email protected] (J. Schäfer).
http://dx.doi.org/10.1016/j.anireprosci.2017.10.014 Received 14 August 2017; Received in revised form 20 October 2017; Accepted 24 October 2017 0378-4320/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Schäfer, J., Animal Reproduction Science (2017), http://dx.doi.org/10.1016/j.anireprosci.2017.10.014
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spermatozoa less susceptible for cold shock (Tamuli and Watson, 1994; Casas and Althouse, 2012). While Yeste et al. (2014) reported an improvement of lots of sperm quality parameters, other studies revealed negative effects on sperm kinematics and fertility when holding time was prolonged from 3 to 24 h (Tomas et al., 2014). A number of other researchers could not detect any differences between varying holding times before freezing (Kong et al., 2012; Gale et al., 2014). Noteworthy, during transport mechanical effects impinge on semen samples. Recently it was shown, that even gentle agitation of liquid preserved semen samples may be harmful to sperm quality (Schulze et al., 2015). Thus, shipping-associated stress factors might influence the response of boar sperm to cooling stress which so far has not been studied.Common freezing protocols contain a further holding step at 5 °C to equilibrate sperm in cooling or freezing extenders at a lower temperature. In bulls, extended equilibration in yolk-extenders at 4–5 °C of about more than 24 h influences sperm quality positively (Griga, 2008; Fleisch et al., 2017) and therefore has become popular in many AI centers. During this period, lipid uptake and metabolism can stabilize plasma membrane structure of spermatozoa (Maldjian et al., 2005; Bergeron and Manjunath, 2006; Svetlichnyy et al., 2014). For boar semen, in general an equilibration period of 2 h is used (Yi et al., 2002). We assume, that this period can be extended when equilibration is done in glycerol-free cooling extender, but not in freezing extender which commonly contains a final concentration of 2–3% glycerol. Thus, spermatozoa will not be exposed to the toxic effects of glycerol for a long period (Fuller, 2004; Macias Garcia et al., 2012; Sieme et al., 2016). At present, the effects of long-term equilibration times in shipped boar semen are not known. The present study was designed to adapt a currently used cryopreservation protocol to pre-freeze conditions associated with predilution and overnight transport at 17 °C. The aims were to evaluate the impact of holding-time of shipped boar semen at 17 °C after arrival in the central laboratory (Experiment 1) and to depict the effect of short- and long-term equilibration in cooling extender at 5 °C (Experiment 2). Overall, it is hypothesized that an adapted protocol offers breeding companies an improved possibility to freeze boar semen in external specialized laboratories. 2. Material and methods 2.1. Chemicals and extenders All chemicals used were of analytical grade. They were purchased from Merck (Darmstadt, Germany), Roth (Karlsruhe, Germany) and Sigma-Aldrich (Taufkirchen, Germany). Transport extender (Beltsville thawing solution, BTS) and thawing extender (Androstar® Premium) were purchased from Minitüb (Tiefenbach, Germany). Cooling extender was composed of 20% egg yolk and 80% lactosesolution (310 mM). Freezing extender consisted of 92.5% cooling extender, 1.5 mL Orvus ES Paste and 6.0 mL glycerol. 2.2. Experimental design Two independent split sample experiments using different ejaculates and random sampling were performed. For each experiment, three single shipments within six weeks in winter took place. The first experiment (Exp. 1, n = 10) was conducted to study the effect of an additional holding time at 17 °C of 1 + 1 (v:v) pre-diluted ejaculates after 20 h of overnight transport to the processing laboratory located at the Institute for the Reproduction of Farm Animals Schönow e.V. (IFN). After arrival, holding time (HT) of semen samples in transport extender was either 2 or 24 h (HT2, HT24). Holding during transport and after arrival in the laboratory summed up to a total holding time of 22 and 44 h, respectively (THT22 and THT44). Direct proceeding after arrival was not done as ejaculates first had to pass quality control, which took about 2 h. The second experiment (Exp. 2, n = 11) was performed to evaluate the influence of short (2 and 4 h) and long (24 and 48 h) equilibration time (ET) in standard lactose-egg yolk cooling extender at 5 °C on boar semen quality post-thaw (ET2, ET4, ET24 and ET48). 2.3. Animals, semen collection and transport Single ejaculates of 21 mature Pietrain boars of proven fertility, aged 1–5 years, from a single boar stud in southern Germany were included in the study. All boars were routinely used for semen production and housed individually in straw-bedded pens according to the European Commission Directives of Pig Welfare. Sperm-rich fraction of the ejaculates was collected once a week using the glovedhand method. Only ejaculates meeting the following requirements for commercial AI were used: semen concentration ≥200 × 106 spermatozoa/mL, raw semen motility ≥70% and ≤25% morphological abnormal spermatozoa. All samples were diluted isothermally 1 + 1 (v:v) with transport extender, filled into QuickTip FlexiTubes® (Minitüb, Tiefenbach, Germany) and manually sealed low-air. Then, they were packed in isolated boxes and sent to the IFN. Transport duration was about 20 h. 2.4. Semen processing Upon arrival, subsamples for semen evaluation were collected from every pre-diluted ejaculate. All samples were split into two (Exp. 1) or four (Exp. 2) groups and put into a climate-controlled device (temperature 17 °C) for the duration of holding time. After holding for 2 or 24 h in Exp. 1 or 2 h in Exp. 2, samples were centrifuged (Rotanta 460R, Hettich, Tuttlingen, Germany) at 17 °C for 3 min at 2400 × g. The supernatant was discarded and semen concentration was adjusted to 1.5 × 109 spermatozoa/mL with cooling extender. Then, samples were placed in a climate-controlled cabinet at 5 °C for 2 h (Exp. 1) or 2, 4, 24 or 48 h (Exp. 2) for equilibration. Subsequently, final dilution with freezing extender to 1.0 × 109 spermatozoa/mL was done. Samples were filled into 0.5 mL medium straws (Minitüb, Germany) using an automated filling device (MPP Uno, Minitüb, Germany). Immediately after filling, 2
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Fig. 1. Freezing protocol for boar semen using IceCube 14S freezing automate (Minitüb, Tiefenbach, Germany). The left side shows the chilling temperature curve; single steps of the program are listed on the right. This protocol was used in both experiments.
samples were transferred into an automated freezing machine (IceCube 14 S, Minitüb, Germany) and frozen as depicted in Fig. 1. Straws were then plunged into liquid nitrogen and stored for at least two weeks before analysis. Four straws of each sample were thawed together at 38 °C for 20 s, using a thawing device with circulating water bath (Geysir, Minitüb, Germany). Straws were emptied into 2.0 mL of pre-warmed thawing extender before further dilution to 20 × 106 spermatozoa/mL for quality control.
2.5. Evaluation of sperm motility Motility was assessed using a computer-assisted semen analysis (CASA) system (AndroVision®, Minitüb, Germany), equipped with a phase contrast microscope (AXIO Scope A1, Carl Zeiss MicroImaging, Göttingen, Germany), a high resolution camera (Basler avA1000-100gc, Basler, Ahrensburg, Germany) and a heated, automatic XY table. Motility analysis post-thawing (PT) was done after 10 min, 30 min and 120 min of incubation in a water bath (Gesellschaft für Labortechnik, Burgwedel, Germany) at 38 °C under air access. Volume of incubated samples was 10 mL. Upon analysis, samples were carefully mixed and 3.0 μL were placed in a previously heated Leja chamber (Leja Products B.V., Nieuw Vennep, The Netherlands). At least 800 spermatozoa per sample were detected and classified according to the manufacturer’s instructions. Spermatozoa were defined motile when showing an amplitude of lateral head displacement (ALH) > 1.0 μm and a velocity curved line (VCL) > 24.0 μm × s−1. When VCL was ≥48.0 μm × s−1 and velocity straight line (VSL) was ≥10.0 μm × s−1, they were listed as progressively motile spermatozoa. 2.6. Flow cytometric assessment of spermatozoa Aliquots for analyses were taken immediately after final dilution PT. Analyses were performed using an Accuri C6 flow cytometer (BD Biosciences, Erembodegem, Belgium) equipped with a 488 nm solid state laser and a 640 nm diode laser. Fluorescence signals of fluorescein-isothiocyanate conjugated peanut agglutinin (FITC-PNA), Pisum sativum agglutinin (FITC-PSA) and rhodamine 123 (R123), gathered via 533/30 nm BP filter, and propidium iodide (PI), gathered via 670 nm LP filter, were plotted on logarithmic scales. Acridine orange (AO), forward- and side-scatter signals were plotted on linear scales. The sperm population was gated referring to the expected forward- and side-scatter signals. A total of 10.000 events in this area were counted. For incubation at 38 °C, a dry block heater was used (Techne Dri-Block® DB2.D, Techne AG, Burkhardtsdorf, Germany).
2.6.1. Evaluation of mitochondrial activity To evaluate mitochondrial activity (MITO), a double-staining with R123 and PI according to Schulze et al. (2013b) was used. In brief, aliquots of 250 μL were mixed with 1.0 μL R123 (final concentration 0.2 μg/mL) and 2.5 μL PI (final concentration 9.9 μg/mL) and incubated under light exclusion for 20 min. After incubation, 15 μL of each sample were mixed with 2.0 mL isotherm phosphatebuffered NaCl solution. The percentage of spermatozoa with active mitochondria and intact plasma membrane was determined.
2.6.2. Evaluation of plasma membrane and acrosome integrity Assessment of plasma membrane and acrosome intact spermatozoa (PMAI) was done by triple-staining with FITC-PNA, FITC-PSA and PI as described previously (Schulze et al., 2013a). First, 125 μL of a fixation solution (containing 0.5% formaldehyde) were supplemented to 375 μL of the sample in lightproof reaction vessels. Then 12.5 μL FITC-PNA and 2.5 μL FITC-PSA (final concentration 2.4 μg/mL each) and, after 5 min of incubation, 5.0 μL PI (final concentration 9.6 μg/mL) were added. Total incubation time at 38 °C was 10 min. Then, 15 μL of the samples were mixed with 2.0 mL isotherm phosphate-buffered NaCl solution. The percentage of sperm with intact plasma and acrosome membrane was determined. 3
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2.6.3. Evaluation of sperm chromatin structure Sperm chromatin structure was analyzed as described by Evenson (Evenson et al., 1994). Briefly, 200 μL TNE-buffered aliquots of semen samples were supplemented with 400 μL of acid solution, mixed on a vortex for 30 s and incubated with 1.2 mL of staining solution (containing AO) on iced water for 3 min in the dark. DNA-fragmentation index (DFI) and high DNA stainability were recorded. 2.7. Temperature control during shipping and equilibration In total, six shipments of semen batches took place. Due to the sensitivity of boar spermatozoa to temperature changes, temperature during shipping and during the first hours of equilibration were recorded in representative samples. For transport, a BL30 data logger (Trotec, Heinzberg, Germany) was used. The temperature probe was placed among 11 semen tubes and temperature was recorded every 5 min from packing until arrival. Arrival temperature of all shipped samples was recorded. To determine the duration and rate of cooling from 17 to 5 °C, an USB-based module (KUSB-3108, Keithley Testpoint 6.1, Keithley Instruments, Ohio, USA) was used. The temperature probe was aligned in the center of a sample (volume 20 mL) containing lactoseegg yolk extender. Temperature of the fluid was recorded for the first 150 min in the climate chamber at 5 °C every 5 min. 2.8. Statistical analysis Data analysis was performed using IBM SPSS Statistics 23 (IBM, Armonk, USA). Data of both experiments failed KolmogorowSmirnow test of normal distribution. For Experiment 1, Wilcoxon signed-rank test was carried out while data of Experiment 2 were analyzed using a Friedman ANOVA on ranks. For post-hoc comparison, Dunn-Bonferroni test was used. Differences were calculated significant when the calculated probability of their occurring by chance was less or equal 5% (P ≤ 0.05). Graphical representation was done with SigmaPlot 13.0 (Systat Software, Erkrath, Germany). Data are presented as median with inter-quartile range (IQR 25–75%). 3. Results 3.1. Experiment 1 There were significant differences in motility and kinematics of thawed spermatozoa between groups THT22 and THT44 (Table 1). Percentage of total and progressively motile spermatozoa (tMot and pMot) differed between THT22 and THT44 (P = 0.013). At 10 min PT there were 37.5% (25.6–43.9%) tMot in THT22 and 33.9% (16.4–43.8%) in THT44. Beat cross frequency (BCF) at 10 min PT also varied between groups to the advantage of THT22 (P = 0.047). At 30 min PT none of the aforementioned differences was confirmed. After 120 min of incubation at 38 °C PT, BCF was higher for THT22 than for THT44 (P = 0.028). There was no statistical significant difference for tMot or pMot at this last measurement. Neither MITO, PMAI nor DFI differed significantly between groups. There was a tendency for better preservation of acrosome and plasma membrane after THT22 in comparison with THT44 (58.7% (42.8–62.0%) vs. 53.1% (45.0–59.9%), P = 0.059). Table 1 Sperm quality parameters with additional holding for 2 or 24 h at 17 °C in transport extender after shipping (Exp. 1, n = 10 ejaculates). Parameters were assessed post-thaw after incubation at 38 °C for 10, 30 and 120 min. Thawing extender was AndroStar® Premium. Final sperm concentration was 20 × 106 spermatozoa x mL−1. Equilibration time at 5 °C was 2 h in both groups. Parameter
PT (min)
HT 2 h
HT 24 h a
tMot (%)
10 30 120
37.5 (25.6–43.9) 34.9 (24.0–43.3)a 17.7 (8.2–29.5)a
33.9 (16.4–43.9)b 35.2 (18.2–45.9)a 19.9 (7.5–25.0)a
pMot (%)
10 30 120
33,9 (24.7–40.7)a 30.8 (23.0–38.8)a 13.0 (8.0–23.8)a
31.5 (13.7–38.9)b 33.0 (15.2–38.9)a 17.1 (5.8–21.2)a
BCF (Hz)
10 30 120
4.84 (1.65–9.78)a 4.36 (1.45–9.32)a 2.11 (0.92–5.19)a
2.79 (1.33–10.10)b 3.61 (1.28–10.61)a 1.69 (0.77–4.36)b
MITO (%) PMAI (%) DFI (%)
10 10 10
40.1 (34.0–45.9)a 58.7 (42.8–62.0)a 1.1 (1.0–1.1)a
39.0 (34.1–43.4)a 53.10 (45.0–59.9)a 1.10 (1.0–1.4)a
All values are medians and inter quartile ranges (25–75%). Abbreviations: PT, post-thaw; HT, holding time; tMot, total motile spermatozoa; pMot, progressive motile spermatozoa; BCF, beat cross frequency; MITO, mitochondrially active spermatozoa; PMAI, plasma membrane and acrosome intact spermatozoa; DFI, DNA fragmentation index. a,b Values with different superscripts within a row differ significantly (P < 0.05).
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Table 2 Sperm quality parameters with equilibration for 2, 4, 24 or 48 h at 5 °C after shipping (Exp. 2, n = 11 ejaculates). Parameters were assessed post-thaw after incubation at 38 °C for 10, 30 and 120 min. Thawing extender was AndroStar® Premium. Final sperm concentration was 20 × 106 spermatozoa x mL−1. Holding time after arrival in laboratory was 2 h in all groups. Parameter
PT (min)
ET 2 h
ET 4 h a
ET 24 h b
ET 48 h a
tMot (%)
10 30 120
48.1 (38.5–51.8) 43.2 (30.9–53.2)ab 37.8 (21.2–40.4)a
52.2 (36.3–55.7) 45.9 (39.9–55.5)a 38.0 (19.4–40.6)a
42.9 (28.2–51.4) 44.6 (30.4–53.5)a,b 24.7 (20.2–37.5)a
37.1 (18.6–49.1)c 39.3 (32.4–42.6)b 19.5 (14.5–25.3)b
pMot (%)
10 30 120
42.0 (34.4–45.8)a 36.7 (28.8–47.6)a 29.3 (17.1–31.7)a
44.0 (31.2–49.1)a 38.7 (31.8–49.3)a 28.2 (16.3–32.3)a
35.4 (21.9–44.8)a 39.4 (26.4–46.9)a 19.6 (15.3–32.4)a
30.5 (14.7–40.6)b 33.1 (28.3–39.1)a 14.3 (10.0–19.3)b
BCF (Hz)
10 30 120
11.27 (6.84–12.64)a 10.64 (6.12–12.16)a 7.40 (4.61–8.17)a
11.88 (7.68–13.37)b 10.65 (8.13–13.04)b 8.34 (4.41–8.60)a
8.79 (5.55–12.65)a 7.86 (6.39–13.11)a,b 5.52 (3.80–7.64)a
6.10 (3.80–9.21)c 7.77 (4.03–9.80)c 3.97 (2.83–5.15)b
MITO (%) PMAI (%) DFI (%)
10 10 10
37.1 (31.3–47.2)a 58.8 (48.3–66.3)a 1.3 (1.1–1.5)a
40.0 (32.0–44.3)a 59.4 (54.8–65.9)a 1.2 (1.1–1.5)a
38.5 (27.4–41.2)a 57.6 (50.6–64.4)a 1.2 (1.0–1.5)a
35.9 (26.5–43.1)a 54.4 (44.7–63.3)a 1.3 (1.1–1.6)a
All values are median and inter quartile range (25–75%). Abbreviations: PT, post-thaw; ET, equilibration time at 5 °C; tMot, total motile spermatozoa; pMot, progressive motile spermatozoa; BCF, beat cross frequency; MITO, mitochondrially active spermatozoa; PMAI, plasma membrane and acrosome intact spermatozoa; DFI, DNA fragmentation index. a–c Values with different superscripts within a row differ significantly (P < 0.05).
3.2. Experiment 2 Motility varied significantly between all groups (Table 2). Best value of tMot at 10 min PT was observed in group ET4 (52.2% (36.3–55.7%)). It was higher than tMot of ET2 (48.1% (38.5–51.8%); P = 0.05), ET24 (42.9% (28.2–51.4%); P = 0.041) and ET48 (37.1% (18.6–49.1%); P = 0.004). Regarding pMot at 10 min PT, groups ET2, ET4 and ET24 displayed significantly higher values than ET48, but did not differ among each other. In BCF at 10 min PT, ET4 was superior to all other groups. There was no statistical difference between groups ET2 and ET24, which were better than ET48 both. At 30 min PT, the only significant difference in tMot was between group ET4 (45.9% (39.9–55.5%)) and ET48 (39.3% (32.4–42.6%); P = 0.026). BCF value of ET48 30 min PT was lower than in all other groups. After 120 min of incubation at 38 °C PT, tMot, pMot and BCF of ET2, ET4 and ET24 statistically did not differ among each other, but were higher than ET48. Differences between groups in MITO, PMAI and DFI were of no significance.
3.3. Temperature profile during transport and equilibration As shown in Fig. 2, during the initial 10 h of transport, temperature of the shipped sample gradually declined from 26.5 to 17.5 °C. Cooling rate was 0.9 °C/h. During further transport, the temperature stayed in the range of 17.5 ± 1.0 °C. Arrival temperature of all shipped samples ranged from 17.4 to 19.6 °C. Fig. 2 shows the temperature decline of a sample that was transferred from a 17 °C cooling chamber to a 5 °C cold store. During the first 20 min, temperature dropped from 17.2 to 12.8 °C (cooling rate 0.22 °C/min). Cooling rate slowed down from 20 to 40 min of equilibration (rate 0.12 °C/min) and dropped below 0.1 °C/min from 40 to 120 min. After 120 min of equilibration, sample
Fig. 2. Temperature profile during shipping of pre-diluted ejaculates (A) to the processing laboratory (batch of 11 samples) and temperature profile of a sample (volume 20 mL, diluted with lactose egg yolk cooling extender) during equilibration in a climate cabinet at 5 °C (B). Temperature profile during shipping and equilibration relate to Exp. 1 and 2.
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temperature was 5.8 °C. 4. Discussion The present study revealed, that pre-diluted boar semen shipped overnight can be cryopreserved with satisfactory outcome if an adapted temperature management is used prior to freezing. Our results show, that sperm quality post-thaw is significantly influenced by holding time after shipment and equilibration time in cooling extender. Earlier studies on the impact of holding and equilibration time on cryopreserved boar semen have been discussed with different results (Polge, 1957; Pursel et al., 1973; Almlid and Johnson, 1988; Eriksson et al., 2001; Yi et al., 2002). Up to now, as per our knowledge neither effect of holding time at 17 °C nor long-term equilibration in cooling extender without glycerol after prior shipping of pre-diluted boar ejaculates have been direct topic of research. Transport of pre-diluted semen in closed insulated boxes assures a slow cooling of samples within the first 12 h after semen collection, thus diminishing chilling injury during the critical temperature range where phase transition of membrane lipids in boar spermatozoa occurs. Influences of prolonged holding time in transport extender at 17 °C after shipment were less pronounced than expected. Results indicate that a total holding time (during transport and after arrival) of 22 h should be preferred to 44 h. This results in higher motility and beat cross frequency 10 min post-thaw. Though, it was noted that these advantages fade with progressing incubation at 38 °C post-thaw. There was a tendency to better acrosome and plasma membrane preservation in the group with the shorter transport and holding time. Other studies indicated an improvement (Casas and Althouse, 2012; Yeste et al., 2014), a deterioration (Guthrie and Welch, 2005) or no effect (Kong et al., 2012; Gale et al., 2014; Tomas et al., 2014) of holding for up to 24 h on sperm quality. In our study, precedent shipment of about 20 h prolonged the period before cryopreservation. Regarding a total transport and holding time of 44 h, it is surprising to what little extend sperm quality post-thaw is diminished after this long period. A pre-dilution 1 + 1 (v:v) with BTS extender proved to avoid aging effects sufficiently for this period. This offers the opportunity to centralize cryopreservation of shipped boar semen in external laboratories even if transport requires more than one day or is accidently delayed. A stable temperature of about 17 °C should be guaranteed and the use of a high quality transport extender might increase safety in this case. Variation of the equilibration time at 5 °C had a stronger influence on post-thaw sperm quality than modification of the holding time. Among the tested durations of equilibration, a period of 4 h resulted in best sperm quality. This implies a doubling of the equilibration time of 2 h, which is mostly used today (Gale et al., 2014; Tomas et al., 2014; Yeste et al., 2014). Prolonging the equilibration time for up to 4 h led to a significant increase in motility and beat cross frequency of frozen-thawed spermatozoa. This is contrary to studies published by Yi et al. (2002). They describe higher sperm motility when 2–3 instead of more hours of equilibration are used. Deviating from our trial, this equilibration time was done in presence of glycerol, which might have a toxic effect on spermatozoa especially in long-term exposition (Fuller, 2004). As glycerol penetrates spermatozoa at 5 °C within a few minutes (Berndtson and Foote, 1972), it is not necessary to do a long equilibration in media containing glycerol to gain its cryoprotective action. Of course, a prolonged equilibration of 4 instead of 2 h at 5 °C also extends total processing time and potential costs for breeders. It has to be evaluated, whether a relatively slight improvement of semen quality justifies this. On the other hand, prolonged equilibration allows more time to perform further tests that ensure high ejaculate quality prior to freezing. Thus, expensive processing and storage of poor quality ejaculates can be avoided more safely. Noteworthy, pre-dilution (Schulze et al., 2013a) and previous cooling regimes influence resistance of spermatozoa to low temperatures. It can be assumed, that slow cooling of pre-diluted semen to 17 °C during transport, as done in the current study, gives better resistance to exposure to 5 °C. A similar effect has been described for buck spermatozoa (Ahmad et al., 2015). Because of the huge time frame between the equilibration times of 4 and 24 h, we cannot state that an equilibration of more than 4, but less than 24 h is of no advance. We did not test these durations, as it would have shifted working into nighttime hours, thereby making it inconvenient for practical use. While equilibration for 24 h at 5 °C is largely not of disadvantage to sperm motility and beat cross frequency compared with 2 h, it is clearly different with 48 h. It can be noticed that boar semen should not be equilibrated in lactoseegg yolk freezing extender for more than 24 h at 5 °C before freezing. This might strongly depend on the extender used, as recently shown for bull spermatozoa (Fleisch et al., 2017), which can be equilibrated for up to 72 h without quality decline. Differences in structure of phospholipid membranes of bull and boar spermatozoa can be one reason for this. Boar spermatozoa membranes have a high phospholipid and low cholesterol content (Parks and Lynch, 1992). This may be disadvantageous for membrane structure changes and lipid metabolism in yolk extenders, which have already been proven also at low temperatures (Cerolini et al., 2001; Svetlichnyy et al., 2014). Despite this, acrosome and plasma membrane integrity was not affected by equilibration time in our study. It can only be speculated that either structure changes have been completed during the first 2 h of equilibration and cooling from 17 to 5 °C or later positive effects were removed due to negative impact of specific yolk components like, for example, high density lipoproteins (Demianowicz and Strzezek, 1995). In addition, other flow cytometric parameters, mitochondria activity and sperm chromatin structure, remained unaffected by the modifications of cryopreservation protocols tested here. Noteworthy, DNA fragmentation in thawed samples remained at low level (DFI less than 2%) confirming the robustness of boar sperm DNA towards freezing technology that has been described previously (Hernandez et al., 2006). Frozen sperm processed from temporarily liquid preserved boar ejaculates therefore will also be useable for advanced assisted reproductive techniques, such as IVF and ICSI. In conclusion, extension of holding time at 17 °C and equilibration period at 5 °C influence the cryopreservation results of overnight-shipped boar semen. Processing of pre-diluted boar semen within 2 h after overnight shipment is preferred, although freezing results are still satisfactory if semen is processed until 44 h after collection. In both cases, equilibration time should be 6
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extended to 4 h. Adaptation of freezing protocols to overnight-shipped semen offers the possibility for high-quality cryopreservation of boar ejaculates to be used in international trade and gene banking. Further studies on the impact of different transport extenders in relation to transport duration and holding time are recommended. Conflict of interest None. Acknowledgments The authors thank Dr. Beate Schumann and Dag-Ronald Bullan for providing boar ejaculates. The technical assistance of Anita Retzlaff and Lutz Rothe is gratefully acknowledged. References Ahmad, M., Nasrullah, R., Ahmad, N., 2015. Effect of cooling rate and equilibration time on pre-freeze and post-thaw survival of buck sperm. Cryobiology 70, 233–238. Almlid, T., Johnson, L.A., 1988. Effects of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa frozen in straws. J. Anim. Sci. 66, 2899–2905. Bergeron, A., Manjunath, P., 2006. New insights towards understanding the mechanisms of sperm protection by egg yolk and milk. Mol. Reprod. Dev. 73, 1338–1344. Berndtson, W.E., Foote, R.H., 1972. The freezability of spermatozoa after minimal pre-freezing exposure to glycerol or lactose. Cryobiology 9, 57–60. Casas, I., Althouse, G.C., 2012. The protective effect of a 17 degrees C holding time on boar sperm plasma membrane fluidity after exposure to 5 degrees C. Cryobiology 66, 69–75. Cerolini, S., Maldjian, A., Pizzi, F., Gliozzi, T.M., 2001. Changes in sperm quality and lipid composition during cryopreservation of boar semen. Reproduction 121, 395–401. Demianowicz, W., Strzezek, J., 1995. The effect of lipoprotein fraction from egg yolk on some of the biological properties of boar spermatozoa during storage of the semen in liquid state. Reprod. Domest. Anim. 31, 279–280. Eriksson, B.M., Vazquez, J.M., Martinez, E.A., Roca, J., Lucas, X., Rodriguez-Martinez, H., 2001. Effects of holding time during cooling and of type of package on plasma membrane integrity, motility and in vitro oocyte penetration ability of frozen-thawed boar spermatozoa. Theriogenology 55, 1593–1605. Evenson, D.P., Thompson, L., Jost, L., 1994. Flow cytometric evaluation of boar semen by the sperm chromatin structure assay as related to cryopreservation and fertility. Theriogenology 41, 637–651. Fleisch, A., Malama, E., Witschi, U., Leiding, C., Siuda, M., Janett, F., Bollwein, H., 2017. Effects of an extension of the equilibration period up to 96 hours on the characteristics of cryopreserved bull semen. Theriogenology 89, 255–262. Fuller, B.J., 2004. Cryoprotectants: the essential antifreezes to protect life in the frozen state. Cryo Lett. 25, 375–388. Gale, I., Gil, L., Malo, C., Gonzalez, N., Martinez, F., 2015. Effect of Camellia sinensis supplementation and increasing holding time on quality of cryopreserved boar semen. Andrologia 47, 505–512. Griga, M.C., 2008. Untersuchungen zur Verbesserung der Technologie der Gefrierkonservierung von Bullensperma. Fachbereich Veterinärmedizin, Freie Universität Berlin, Berlin, pp. 149. Guthrie, H.D., Welch, G.R., 2005. Impact of storage prior to cryopreservation on plasma membrane function and fertility of boar sperm. Theriogenology 63, 396–410. Hernandez, M., Roca, J., Ballester, J., Vazquez, J.M., Martinez, E.A., Johannisson, A., Saravia, F., Rodriguez-Martinez, H., 2006. Differences in SCSA outcome among boars with different sperm freezability. Int. J. Androl. 29, 583–591. Knox, R.V., 2016. Artificial insemination in pigs today. Theriogenology 85, 83–93. Kong, D., Shang, H., Guo, K., Liu, Y., Zhang, J., Wei, H., 2012. A study on optimizing the cryopreservation methods for Bama miniature pig semen. Exp. Anim. 61, 533–542. Macias Garcia, B., Ortega Ferrusola, C., Aparicio, I.M., Miro-Moran, A., Morillo Rodriguez, A., Gallardo Bolanos, J.M., Gonzalez Fernandez, L., Balaoda Silva, C.M., Rodriguez Martinez, H., Tapia, J.A., Pena, F.J., 2012. Toxicity of glycerol for the stallion spermatozoa: effects on membrane integrity and cytoskeleton, lipid peroxidation and mitochondrial membrane potential. Theriogenology 77, 1280–1289. Maldjian, A., Pizzi, F., Gliozzi, T., Cerolini, S., Penny, P., Noble, R., 2005. Changes in sperm quality and lipid composition during cryopreservation of boar semen. Theriogenology 63, 411–421. Maxwell, W.M., Johnson, L.A., 1997. Membrane status of boar spermatozoa after cooling or cryopreservation. Theriogenology 48, 209–219. Men, H., Walters, E.M., Nagashima, H., Prather, R.S., 2012. Emerging applications of sperm, embryo and somatic cell cryopreservation in maintenance, relocation and rederivation of swine genetics. Theriogenology 78, 1720–1729. Parks, J.E., Lynch, D.V., 1992. Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes. Cryobiology 29, 255–266. Polge, C., 1957. Low-temperature storage of mammalian spermatozoa. Series B, Biological Sciences. Royal Society of London, London, pp. 498–508. Pursel, V.G., Schulman, L.L., Johnson, L.A., 1973. Effect of holding time on storage of boar spermatozoa at 5C. J. Anim. Sci. 37, 785–789. Rodríguez-Gil, J.E., Estrada, E., 2013. Artificial insemination in boar reproduction. In: Bonet, S., Casas, I., Holt, W.V., Yeste, M. (Eds.), Boar Reproduction: Fundamentals and New Biotechnological Trends. Springer Berlin Heidelberg, Berlin, Heidelberg, pp. 589–607. Schulze, M., Henning, H., Rüdiger, K., Wallner, U., Waberski, D., 2013a. Temperature management during semen processing: impact on boar sperm quality under laboratory and field conditions. Theriogenology 80, 990–998. Schulze, M., Rüdiger, K., Müller, K., Jung, M., Well, C., Reissmann, M., 2013b. Development of an in vitro index to characterize fertilizing capacity of boar ejaculates. Anim. Reprod. Sci. 140, 70–76. Schulze, M., Rüdiger, K., Waberski, D., 2015. Rotation of boar semen doses during storage affects sperm quality. Reprod. Domest. Anim. 50, 684–687. Sieme, H., Oldenhof, H., Wolkers, W.F., 2016. Mode of action of cryoprotectants for sperm preservation. Anim. Reprod. Sci. 169, 2–5. Svetlichnyy, V., Müller, P., Pomorski, T.G., Schulze, M., Schiller, J., Müller, K., 2014. Metabolic incorporation of unsaturated fatty acids into boar spermatozoa lipids and de novo formation of diacylglycerols. Chem. Phys. Lipids 177, 41–50. Tamuli, M.K., Watson, P.F., 1994. Cold resistance of live boar spermatozoa during incubation after ejaculation. Vet. Rec. 135, 160–162. Tomas, C., Gomez-Fernandez, J., Gomez-Izquierdo, E., de Mercado, E., 2014. Effect of the holding time at 15 degrees C prior to cryopreservation, the thawing rate and the post-thaw incubation temperature on the boar sperm quality after cryopreservation. Anim. Reprod. Sci. 144, 115–121. Yeste, M., Estrada, E., Rivera Del Alamo, M.M., Bonet, S., Rigau, T., Rodriguez-Gil, J.E., 2014. The increase in phosphorylation levels of serine residues of protein HSP70 during holding time at 17 degrees C is concomitant with a higher cryotolerance of boar spermatozoa. PLoS One 9, e90887. Yi, Y.J., Cheon, Y.M., Park, C.S., 2002. Effect of N-acetyl-D-glucosamine, and glycerol concentration and equilibration time on acrosome morphology and motility of frozen-thawed boar sperm. Anim. Reprod. Sci. 69, 91–97.
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Impact of holding and equilibration time on post-thaw quality of shipped boar semen ⁎
J. Schäfera,b, , D. Waberskib, M. Junga, M. Schulzea a b
Institute for the Reproduction of Farm Animals Schönow e.V. (IFN), Bernauer Allee 10, D-16321 Bernau, Germany Unit for Reproductive Medicine of Clinics/Clinic for Pigs and Small Ruminants, University of Veterinary Medicine Hannover, Hannover, Germany
AR TI CLE I NF O
AB S T R A CT
Keywords: Boar semen Cryopreservation Equilibration Holding time Shipping
Cryopreservation of boar semen is of growing interest for breeding companies. Overnight-shipping of pre-diluted ejaculates to specialized laboratories offers a practicable method, but requires fine-tuned protocols. In this study, the impact of holding post shipping at 17 °C for 2 or 24 h (n = 10 samples) and of equilibration in lactose-egg yolk extender without glycerol at 5 °C for 2, 4, 24 or 48 h (n = 11 samples) before freezing was investigated. Sperm-rich fractions of ejaculates from 21 mature Pietrain boars were collected at a single boar stud. After pre-dilution (1 + 1, v:v) with Beltsville thawing solution, samples were sent to the laboratory. Temperature profiles during transport and initial equilibration time were recorded. Semen quality post-thaw (PT) was evaluated using CASA and flow cytometry. Holding of 2 h after shipping resulted in higher sperm motility (P = 0.013) and beat cross frequency (BCF; P = 0.047) compared to 24 h. Differences between both groups vanished with prolonged incubation at 38 °C PT. Equilibration at 5 °C for 4 h yielded the highest motility and BCF, whereas the equilibration for 48 h impaired sperm motility. Membrane integrity, mitochondrial activity and DNA fragmentation index were not affected by any protocol modification. In conclusion, processing of pre-diluted boar semen shipped overnight within 2 h after arrival at the laboratory is preferred to 24 h of additional holding at 17 °C. Extending the equilibration period in lactose-egg yolk extender without glycerol at 5 °C from 2 h to 4 h before freezing is recommended.
1. Introduction Demand on freezing valuable boars’ ejaculates for international trade and gene banking is high (Men et al., 2012; Knox, 2016). Worldwide less than 1% artificial inseminations (AI) in pigs are performed with frozen-thawed semen (Rodríguez-Gil and Estrada, 2013). Due to the composition of sperm membranes with high protein to phospholipid and low cholesterol to phospholipid ratios, boar spermatozoa are highly sensitive to low temperatures. Chilling injury is expressed as loss of motility, membrane integrity, mitochondrial activity and other sperm functions (Parks and Lynch, 1992; Maxwell and Johnson, 1997). Cryopreservation protocols for boar semen are delicate and complex in order to minimize sperm damage during processing and consequently are often not practicable in the daily routine of AI stations. Shipping of pre-diluted ejaculates to a central personally and technically well-equipped laboratory would allow freezing under ideal conditions thus offering a feasible, cost-efficient method for breeding companies. Overnight shipping inevitably introduces a holding time of extended semen for approximately 24 h at 17 °C before further processing. The effects of holding time at 15–17 °C prior to cryopreservation have been discussed with different outcomes. It is assumed, that during holding time an alteration of the plasma membrane lipid composition and protein phosphorylation takes place, rendering the ⁎
Corresponding author at: Institute for the Reproduction of Farm Animals Schönow e.V. (IFN), Bernauer Allee 10, D-16321 Bernau, Germany. E-mail address: [email protected] (J. Schäfer).
http://dx.doi.org/10.1016/j.anireprosci.2017.10.014 Received 14 August 2017; Received in revised form 20 October 2017; Accepted 24 October 2017 0378-4320/ © 2017 Elsevier B.V. All rights reserved.
Please cite this article as: Schäfer, J., Animal Reproduction Science (2017), http://dx.doi.org/10.1016/j.anireprosci.2017.10.014
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spermatozoa less susceptible for cold shock (Tamuli and Watson, 1994; Casas and Althouse, 2012). While Yeste et al. (2014) reported an improvement of lots of sperm quality parameters, other studies revealed negative effects on sperm kinematics and fertility when holding time was prolonged from 3 to 24 h (Tomas et al., 2014). A number of other researchers could not detect any differences between varying holding times before freezing (Kong et al., 2012; Gale et al., 2014). Noteworthy, during transport mechanical effects impinge on semen samples. Recently it was shown, that even gentle agitation of liquid preserved semen samples may be harmful to sperm quality (Schulze et al., 2015). Thus, shipping-associated stress factors might influence the response of boar sperm to cooling stress which so far has not been studied.Common freezing protocols contain a further holding step at 5 °C to equilibrate sperm in cooling or freezing extenders at a lower temperature. In bulls, extended equilibration in yolk-extenders at 4–5 °C of about more than 24 h influences sperm quality positively (Griga, 2008; Fleisch et al., 2017) and therefore has become popular in many AI centers. During this period, lipid uptake and metabolism can stabilize plasma membrane structure of spermatozoa (Maldjian et al., 2005; Bergeron and Manjunath, 2006; Svetlichnyy et al., 2014). For boar semen, in general an equilibration period of 2 h is used (Yi et al., 2002). We assume, that this period can be extended when equilibration is done in glycerol-free cooling extender, but not in freezing extender which commonly contains a final concentration of 2–3% glycerol. Thus, spermatozoa will not be exposed to the toxic effects of glycerol for a long period (Fuller, 2004; Macias Garcia et al., 2012; Sieme et al., 2016). At present, the effects of long-term equilibration times in shipped boar semen are not known. The present study was designed to adapt a currently used cryopreservation protocol to pre-freeze conditions associated with predilution and overnight transport at 17 °C. The aims were to evaluate the impact of holding-time of shipped boar semen at 17 °C after arrival in the central laboratory (Experiment 1) and to depict the effect of short- and long-term equilibration in cooling extender at 5 °C (Experiment 2). Overall, it is hypothesized that an adapted protocol offers breeding companies an improved possibility to freeze boar semen in external specialized laboratories. 2. Material and methods 2.1. Chemicals and extenders All chemicals used were of analytical grade. They were purchased from Merck (Darmstadt, Germany), Roth (Karlsruhe, Germany) and Sigma-Aldrich (Taufkirchen, Germany). Transport extender (Beltsville thawing solution, BTS) and thawing extender (Androstar® Premium) were purchased from Minitüb (Tiefenbach, Germany). Cooling extender was composed of 20% egg yolk and 80% lactosesolution (310 mM). Freezing extender consisted of 92.5% cooling extender, 1.5 mL Orvus ES Paste and 6.0 mL glycerol. 2.2. Experimental design Two independent split sample experiments using different ejaculates and random sampling were performed. For each experiment, three single shipments within six weeks in winter took place. The first experiment (Exp. 1, n = 10) was conducted to study the effect of an additional holding time at 17 °C of 1 + 1 (v:v) pre-diluted ejaculates after 20 h of overnight transport to the processing laboratory located at the Institute for the Reproduction of Farm Animals Schönow e.V. (IFN). After arrival, holding time (HT) of semen samples in transport extender was either 2 or 24 h (HT2, HT24). Holding during transport and after arrival in the laboratory summed up to a total holding time of 22 and 44 h, respectively (THT22 and THT44). Direct proceeding after arrival was not done as ejaculates first had to pass quality control, which took about 2 h. The second experiment (Exp. 2, n = 11) was performed to evaluate the influence of short (2 and 4 h) and long (24 and 48 h) equilibration time (ET) in standard lactose-egg yolk cooling extender at 5 °C on boar semen quality post-thaw (ET2, ET4, ET24 and ET48). 2.3. Animals, semen collection and transport Single ejaculates of 21 mature Pietrain boars of proven fertility, aged 1–5 years, from a single boar stud in southern Germany were included in the study. All boars were routinely used for semen production and housed individually in straw-bedded pens according to the European Commission Directives of Pig Welfare. Sperm-rich fraction of the ejaculates was collected once a week using the glovedhand method. Only ejaculates meeting the following requirements for commercial AI were used: semen concentration ≥200 × 106 spermatozoa/mL, raw semen motility ≥70% and ≤25% morphological abnormal spermatozoa. All samples were diluted isothermally 1 + 1 (v:v) with transport extender, filled into QuickTip FlexiTubes® (Minitüb, Tiefenbach, Germany) and manually sealed low-air. Then, they were packed in isolated boxes and sent to the IFN. Transport duration was about 20 h. 2.4. Semen processing Upon arrival, subsamples for semen evaluation were collected from every pre-diluted ejaculate. All samples were split into two (Exp. 1) or four (Exp. 2) groups and put into a climate-controlled device (temperature 17 °C) for the duration of holding time. After holding for 2 or 24 h in Exp. 1 or 2 h in Exp. 2, samples were centrifuged (Rotanta 460R, Hettich, Tuttlingen, Germany) at 17 °C for 3 min at 2400 × g. The supernatant was discarded and semen concentration was adjusted to 1.5 × 109 spermatozoa/mL with cooling extender. Then, samples were placed in a climate-controlled cabinet at 5 °C for 2 h (Exp. 1) or 2, 4, 24 or 48 h (Exp. 2) for equilibration. Subsequently, final dilution with freezing extender to 1.0 × 109 spermatozoa/mL was done. Samples were filled into 0.5 mL medium straws (Minitüb, Germany) using an automated filling device (MPP Uno, Minitüb, Germany). Immediately after filling, 2
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Fig. 1. Freezing protocol for boar semen using IceCube 14S freezing automate (Minitüb, Tiefenbach, Germany). The left side shows the chilling temperature curve; single steps of the program are listed on the right. This protocol was used in both experiments.
samples were transferred into an automated freezing machine (IceCube 14 S, Minitüb, Germany) and frozen as depicted in Fig. 1. Straws were then plunged into liquid nitrogen and stored for at least two weeks before analysis. Four straws of each sample were thawed together at 38 °C for 20 s, using a thawing device with circulating water bath (Geysir, Minitüb, Germany). Straws were emptied into 2.0 mL of pre-warmed thawing extender before further dilution to 20 × 106 spermatozoa/mL for quality control.
2.5. Evaluation of sperm motility Motility was assessed using a computer-assisted semen analysis (CASA) system (AndroVision®, Minitüb, Germany), equipped with a phase contrast microscope (AXIO Scope A1, Carl Zeiss MicroImaging, Göttingen, Germany), a high resolution camera (Basler avA1000-100gc, Basler, Ahrensburg, Germany) and a heated, automatic XY table. Motility analysis post-thawing (PT) was done after 10 min, 30 min and 120 min of incubation in a water bath (Gesellschaft für Labortechnik, Burgwedel, Germany) at 38 °C under air access. Volume of incubated samples was 10 mL. Upon analysis, samples were carefully mixed and 3.0 μL were placed in a previously heated Leja chamber (Leja Products B.V., Nieuw Vennep, The Netherlands). At least 800 spermatozoa per sample were detected and classified according to the manufacturer’s instructions. Spermatozoa were defined motile when showing an amplitude of lateral head displacement (ALH) > 1.0 μm and a velocity curved line (VCL) > 24.0 μm × s−1. When VCL was ≥48.0 μm × s−1 and velocity straight line (VSL) was ≥10.0 μm × s−1, they were listed as progressively motile spermatozoa. 2.6. Flow cytometric assessment of spermatozoa Aliquots for analyses were taken immediately after final dilution PT. Analyses were performed using an Accuri C6 flow cytometer (BD Biosciences, Erembodegem, Belgium) equipped with a 488 nm solid state laser and a 640 nm diode laser. Fluorescence signals of fluorescein-isothiocyanate conjugated peanut agglutinin (FITC-PNA), Pisum sativum agglutinin (FITC-PSA) and rhodamine 123 (R123), gathered via 533/30 nm BP filter, and propidium iodide (PI), gathered via 670 nm LP filter, were plotted on logarithmic scales. Acridine orange (AO), forward- and side-scatter signals were plotted on linear scales. The sperm population was gated referring to the expected forward- and side-scatter signals. A total of 10.000 events in this area were counted. For incubation at 38 °C, a dry block heater was used (Techne Dri-Block® DB2.D, Techne AG, Burkhardtsdorf, Germany).
2.6.1. Evaluation of mitochondrial activity To evaluate mitochondrial activity (MITO), a double-staining with R123 and PI according to Schulze et al. (2013b) was used. In brief, aliquots of 250 μL were mixed with 1.0 μL R123 (final concentration 0.2 μg/mL) and 2.5 μL PI (final concentration 9.9 μg/mL) and incubated under light exclusion for 20 min. After incubation, 15 μL of each sample were mixed with 2.0 mL isotherm phosphatebuffered NaCl solution. The percentage of spermatozoa with active mitochondria and intact plasma membrane was determined.
2.6.2. Evaluation of plasma membrane and acrosome integrity Assessment of plasma membrane and acrosome intact spermatozoa (PMAI) was done by triple-staining with FITC-PNA, FITC-PSA and PI as described previously (Schulze et al., 2013a). First, 125 μL of a fixation solution (containing 0.5% formaldehyde) were supplemented to 375 μL of the sample in lightproof reaction vessels. Then 12.5 μL FITC-PNA and 2.5 μL FITC-PSA (final concentration 2.4 μg/mL each) and, after 5 min of incubation, 5.0 μL PI (final concentration 9.6 μg/mL) were added. Total incubation time at 38 °C was 10 min. Then, 15 μL of the samples were mixed with 2.0 mL isotherm phosphate-buffered NaCl solution. The percentage of sperm with intact plasma and acrosome membrane was determined. 3
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2.6.3. Evaluation of sperm chromatin structure Sperm chromatin structure was analyzed as described by Evenson (Evenson et al., 1994). Briefly, 200 μL TNE-buffered aliquots of semen samples were supplemented with 400 μL of acid solution, mixed on a vortex for 30 s and incubated with 1.2 mL of staining solution (containing AO) on iced water for 3 min in the dark. DNA-fragmentation index (DFI) and high DNA stainability were recorded. 2.7. Temperature control during shipping and equilibration In total, six shipments of semen batches took place. Due to the sensitivity of boar spermatozoa to temperature changes, temperature during shipping and during the first hours of equilibration were recorded in representative samples. For transport, a BL30 data logger (Trotec, Heinzberg, Germany) was used. The temperature probe was placed among 11 semen tubes and temperature was recorded every 5 min from packing until arrival. Arrival temperature of all shipped samples was recorded. To determine the duration and rate of cooling from 17 to 5 °C, an USB-based module (KUSB-3108, Keithley Testpoint 6.1, Keithley Instruments, Ohio, USA) was used. The temperature probe was aligned in the center of a sample (volume 20 mL) containing lactoseegg yolk extender. Temperature of the fluid was recorded for the first 150 min in the climate chamber at 5 °C every 5 min. 2.8. Statistical analysis Data analysis was performed using IBM SPSS Statistics 23 (IBM, Armonk, USA). Data of both experiments failed KolmogorowSmirnow test of normal distribution. For Experiment 1, Wilcoxon signed-rank test was carried out while data of Experiment 2 were analyzed using a Friedman ANOVA on ranks. For post-hoc comparison, Dunn-Bonferroni test was used. Differences were calculated significant when the calculated probability of their occurring by chance was less or equal 5% (P ≤ 0.05). Graphical representation was done with SigmaPlot 13.0 (Systat Software, Erkrath, Germany). Data are presented as median with inter-quartile range (IQR 25–75%). 3. Results 3.1. Experiment 1 There were significant differences in motility and kinematics of thawed spermatozoa between groups THT22 and THT44 (Table 1). Percentage of total and progressively motile spermatozoa (tMot and pMot) differed between THT22 and THT44 (P = 0.013). At 10 min PT there were 37.5% (25.6–43.9%) tMot in THT22 and 33.9% (16.4–43.8%) in THT44. Beat cross frequency (BCF) at 10 min PT also varied between groups to the advantage of THT22 (P = 0.047). At 30 min PT none of the aforementioned differences was confirmed. After 120 min of incubation at 38 °C PT, BCF was higher for THT22 than for THT44 (P = 0.028). There was no statistical significant difference for tMot or pMot at this last measurement. Neither MITO, PMAI nor DFI differed significantly between groups. There was a tendency for better preservation of acrosome and plasma membrane after THT22 in comparison with THT44 (58.7% (42.8–62.0%) vs. 53.1% (45.0–59.9%), P = 0.059). Table 1 Sperm quality parameters with additional holding for 2 or 24 h at 17 °C in transport extender after shipping (Exp. 1, n = 10 ejaculates). Parameters were assessed post-thaw after incubation at 38 °C for 10, 30 and 120 min. Thawing extender was AndroStar® Premium. Final sperm concentration was 20 × 106 spermatozoa x mL−1. Equilibration time at 5 °C was 2 h in both groups. Parameter
PT (min)
HT 2 h
HT 24 h a
tMot (%)
10 30 120
37.5 (25.6–43.9) 34.9 (24.0–43.3)a 17.7 (8.2–29.5)a
33.9 (16.4–43.9)b 35.2 (18.2–45.9)a 19.9 (7.5–25.0)a
pMot (%)
10 30 120
33,9 (24.7–40.7)a 30.8 (23.0–38.8)a 13.0 (8.0–23.8)a
31.5 (13.7–38.9)b 33.0 (15.2–38.9)a 17.1 (5.8–21.2)a
BCF (Hz)
10 30 120
4.84 (1.65–9.78)a 4.36 (1.45–9.32)a 2.11 (0.92–5.19)a
2.79 (1.33–10.10)b 3.61 (1.28–10.61)a 1.69 (0.77–4.36)b
MITO (%) PMAI (%) DFI (%)
10 10 10
40.1 (34.0–45.9)a 58.7 (42.8–62.0)a 1.1 (1.0–1.1)a
39.0 (34.1–43.4)a 53.10 (45.0–59.9)a 1.10 (1.0–1.4)a
All values are medians and inter quartile ranges (25–75%). Abbreviations: PT, post-thaw; HT, holding time; tMot, total motile spermatozoa; pMot, progressive motile spermatozoa; BCF, beat cross frequency; MITO, mitochondrially active spermatozoa; PMAI, plasma membrane and acrosome intact spermatozoa; DFI, DNA fragmentation index. a,b Values with different superscripts within a row differ significantly (P < 0.05).
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Table 2 Sperm quality parameters with equilibration for 2, 4, 24 or 48 h at 5 °C after shipping (Exp. 2, n = 11 ejaculates). Parameters were assessed post-thaw after incubation at 38 °C for 10, 30 and 120 min. Thawing extender was AndroStar® Premium. Final sperm concentration was 20 × 106 spermatozoa x mL−1. Holding time after arrival in laboratory was 2 h in all groups. Parameter
PT (min)
ET 2 h
ET 4 h a
ET 24 h b
ET 48 h a
tMot (%)
10 30 120
48.1 (38.5–51.8) 43.2 (30.9–53.2)ab 37.8 (21.2–40.4)a
52.2 (36.3–55.7) 45.9 (39.9–55.5)a 38.0 (19.4–40.6)a
42.9 (28.2–51.4) 44.6 (30.4–53.5)a,b 24.7 (20.2–37.5)a
37.1 (18.6–49.1)c 39.3 (32.4–42.6)b 19.5 (14.5–25.3)b
pMot (%)
10 30 120
42.0 (34.4–45.8)a 36.7 (28.8–47.6)a 29.3 (17.1–31.7)a
44.0 (31.2–49.1)a 38.7 (31.8–49.3)a 28.2 (16.3–32.3)a
35.4 (21.9–44.8)a 39.4 (26.4–46.9)a 19.6 (15.3–32.4)a
30.5 (14.7–40.6)b 33.1 (28.3–39.1)a 14.3 (10.0–19.3)b
BCF (Hz)
10 30 120
11.27 (6.84–12.64)a 10.64 (6.12–12.16)a 7.40 (4.61–8.17)a
11.88 (7.68–13.37)b 10.65 (8.13–13.04)b 8.34 (4.41–8.60)a
8.79 (5.55–12.65)a 7.86 (6.39–13.11)a,b 5.52 (3.80–7.64)a
6.10 (3.80–9.21)c 7.77 (4.03–9.80)c 3.97 (2.83–5.15)b
MITO (%) PMAI (%) DFI (%)
10 10 10
37.1 (31.3–47.2)a 58.8 (48.3–66.3)a 1.3 (1.1–1.5)a
40.0 (32.0–44.3)a 59.4 (54.8–65.9)a 1.2 (1.1–1.5)a
38.5 (27.4–41.2)a 57.6 (50.6–64.4)a 1.2 (1.0–1.5)a
35.9 (26.5–43.1)a 54.4 (44.7–63.3)a 1.3 (1.1–1.6)a
All values are median and inter quartile range (25–75%). Abbreviations: PT, post-thaw; ET, equilibration time at 5 °C; tMot, total motile spermatozoa; pMot, progressive motile spermatozoa; BCF, beat cross frequency; MITO, mitochondrially active spermatozoa; PMAI, plasma membrane and acrosome intact spermatozoa; DFI, DNA fragmentation index. a–c Values with different superscripts within a row differ significantly (P < 0.05).
3.2. Experiment 2 Motility varied significantly between all groups (Table 2). Best value of tMot at 10 min PT was observed in group ET4 (52.2% (36.3–55.7%)). It was higher than tMot of ET2 (48.1% (38.5–51.8%); P = 0.05), ET24 (42.9% (28.2–51.4%); P = 0.041) and ET48 (37.1% (18.6–49.1%); P = 0.004). Regarding pMot at 10 min PT, groups ET2, ET4 and ET24 displayed significantly higher values than ET48, but did not differ among each other. In BCF at 10 min PT, ET4 was superior to all other groups. There was no statistical difference between groups ET2 and ET24, which were better than ET48 both. At 30 min PT, the only significant difference in tMot was between group ET4 (45.9% (39.9–55.5%)) and ET48 (39.3% (32.4–42.6%); P = 0.026). BCF value of ET48 30 min PT was lower than in all other groups. After 120 min of incubation at 38 °C PT, tMot, pMot and BCF of ET2, ET4 and ET24 statistically did not differ among each other, but were higher than ET48. Differences between groups in MITO, PMAI and DFI were of no significance.
3.3. Temperature profile during transport and equilibration As shown in Fig. 2, during the initial 10 h of transport, temperature of the shipped sample gradually declined from 26.5 to 17.5 °C. Cooling rate was 0.9 °C/h. During further transport, the temperature stayed in the range of 17.5 ± 1.0 °C. Arrival temperature of all shipped samples ranged from 17.4 to 19.6 °C. Fig. 2 shows the temperature decline of a sample that was transferred from a 17 °C cooling chamber to a 5 °C cold store. During the first 20 min, temperature dropped from 17.2 to 12.8 °C (cooling rate 0.22 °C/min). Cooling rate slowed down from 20 to 40 min of equilibration (rate 0.12 °C/min) and dropped below 0.1 °C/min from 40 to 120 min. After 120 min of equilibration, sample
Fig. 2. Temperature profile during shipping of pre-diluted ejaculates (A) to the processing laboratory (batch of 11 samples) and temperature profile of a sample (volume 20 mL, diluted with lactose egg yolk cooling extender) during equilibration in a climate cabinet at 5 °C (B). Temperature profile during shipping and equilibration relate to Exp. 1 and 2.
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temperature was 5.8 °C. 4. Discussion The present study revealed, that pre-diluted boar semen shipped overnight can be cryopreserved with satisfactory outcome if an adapted temperature management is used prior to freezing. Our results show, that sperm quality post-thaw is significantly influenced by holding time after shipment and equilibration time in cooling extender. Earlier studies on the impact of holding and equilibration time on cryopreserved boar semen have been discussed with different results (Polge, 1957; Pursel et al., 1973; Almlid and Johnson, 1988; Eriksson et al., 2001; Yi et al., 2002). Up to now, as per our knowledge neither effect of holding time at 17 °C nor long-term equilibration in cooling extender without glycerol after prior shipping of pre-diluted boar ejaculates have been direct topic of research. Transport of pre-diluted semen in closed insulated boxes assures a slow cooling of samples within the first 12 h after semen collection, thus diminishing chilling injury during the critical temperature range where phase transition of membrane lipids in boar spermatozoa occurs. Influences of prolonged holding time in transport extender at 17 °C after shipment were less pronounced than expected. Results indicate that a total holding time (during transport and after arrival) of 22 h should be preferred to 44 h. This results in higher motility and beat cross frequency 10 min post-thaw. Though, it was noted that these advantages fade with progressing incubation at 38 °C post-thaw. There was a tendency to better acrosome and plasma membrane preservation in the group with the shorter transport and holding time. Other studies indicated an improvement (Casas and Althouse, 2012; Yeste et al., 2014), a deterioration (Guthrie and Welch, 2005) or no effect (Kong et al., 2012; Gale et al., 2014; Tomas et al., 2014) of holding for up to 24 h on sperm quality. In our study, precedent shipment of about 20 h prolonged the period before cryopreservation. Regarding a total transport and holding time of 44 h, it is surprising to what little extend sperm quality post-thaw is diminished after this long period. A pre-dilution 1 + 1 (v:v) with BTS extender proved to avoid aging effects sufficiently for this period. This offers the opportunity to centralize cryopreservation of shipped boar semen in external laboratories even if transport requires more than one day or is accidently delayed. A stable temperature of about 17 °C should be guaranteed and the use of a high quality transport extender might increase safety in this case. Variation of the equilibration time at 5 °C had a stronger influence on post-thaw sperm quality than modification of the holding time. Among the tested durations of equilibration, a period of 4 h resulted in best sperm quality. This implies a doubling of the equilibration time of 2 h, which is mostly used today (Gale et al., 2014; Tomas et al., 2014; Yeste et al., 2014). Prolonging the equilibration time for up to 4 h led to a significant increase in motility and beat cross frequency of frozen-thawed spermatozoa. This is contrary to studies published by Yi et al. (2002). They describe higher sperm motility when 2–3 instead of more hours of equilibration are used. Deviating from our trial, this equilibration time was done in presence of glycerol, which might have a toxic effect on spermatozoa especially in long-term exposition (Fuller, 2004). As glycerol penetrates spermatozoa at 5 °C within a few minutes (Berndtson and Foote, 1972), it is not necessary to do a long equilibration in media containing glycerol to gain its cryoprotective action. Of course, a prolonged equilibration of 4 instead of 2 h at 5 °C also extends total processing time and potential costs for breeders. It has to be evaluated, whether a relatively slight improvement of semen quality justifies this. On the other hand, prolonged equilibration allows more time to perform further tests that ensure high ejaculate quality prior to freezing. Thus, expensive processing and storage of poor quality ejaculates can be avoided more safely. Noteworthy, pre-dilution (Schulze et al., 2013a) and previous cooling regimes influence resistance of spermatozoa to low temperatures. It can be assumed, that slow cooling of pre-diluted semen to 17 °C during transport, as done in the current study, gives better resistance to exposure to 5 °C. A similar effect has been described for buck spermatozoa (Ahmad et al., 2015). Because of the huge time frame between the equilibration times of 4 and 24 h, we cannot state that an equilibration of more than 4, but less than 24 h is of no advance. We did not test these durations, as it would have shifted working into nighttime hours, thereby making it inconvenient for practical use. While equilibration for 24 h at 5 °C is largely not of disadvantage to sperm motility and beat cross frequency compared with 2 h, it is clearly different with 48 h. It can be noticed that boar semen should not be equilibrated in lactoseegg yolk freezing extender for more than 24 h at 5 °C before freezing. This might strongly depend on the extender used, as recently shown for bull spermatozoa (Fleisch et al., 2017), which can be equilibrated for up to 72 h without quality decline. Differences in structure of phospholipid membranes of bull and boar spermatozoa can be one reason for this. Boar spermatozoa membranes have a high phospholipid and low cholesterol content (Parks and Lynch, 1992). This may be disadvantageous for membrane structure changes and lipid metabolism in yolk extenders, which have already been proven also at low temperatures (Cerolini et al., 2001; Svetlichnyy et al., 2014). Despite this, acrosome and plasma membrane integrity was not affected by equilibration time in our study. It can only be speculated that either structure changes have been completed during the first 2 h of equilibration and cooling from 17 to 5 °C or later positive effects were removed due to negative impact of specific yolk components like, for example, high density lipoproteins (Demianowicz and Strzezek, 1995). In addition, other flow cytometric parameters, mitochondria activity and sperm chromatin structure, remained unaffected by the modifications of cryopreservation protocols tested here. Noteworthy, DNA fragmentation in thawed samples remained at low level (DFI less than 2%) confirming the robustness of boar sperm DNA towards freezing technology that has been described previously (Hernandez et al., 2006). Frozen sperm processed from temporarily liquid preserved boar ejaculates therefore will also be useable for advanced assisted reproductive techniques, such as IVF and ICSI. In conclusion, extension of holding time at 17 °C and equilibration period at 5 °C influence the cryopreservation results of overnight-shipped boar semen. Processing of pre-diluted boar semen within 2 h after overnight shipment is preferred, although freezing results are still satisfactory if semen is processed until 44 h after collection. In both cases, equilibration time should be 6
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extended to 4 h. Adaptation of freezing protocols to overnight-shipped semen offers the possibility for high-quality cryopreservation of boar ejaculates to be used in international trade and gene banking. Further studies on the impact of different transport extenders in relation to transport duration and holding time are recommended. Conflict of interest None. Acknowledgments The authors thank Dr. Beate Schumann and Dag-Ronald Bullan for providing boar ejaculates. The technical assistance of Anita Retzlaff and Lutz Rothe is gratefully acknowledged. References Ahmad, M., Nasrullah, R., Ahmad, N., 2015. Effect of cooling rate and equilibration time on pre-freeze and post-thaw survival of buck sperm. Cryobiology 70, 233–238. Almlid, T., Johnson, L.A., 1988. Effects of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa frozen in straws. J. Anim. Sci. 66, 2899–2905. Bergeron, A., Manjunath, P., 2006. 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