Cryobiology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury, but respond favorably to dimethyl sulfoxide q,qq Amy E.M. Johnson a,b, Elizabeth W. Freeman c, David E. Wildt a, Nucharin Songsasen a,⇑ a b c
Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA, USA Department of Environmental Science and Policy, George Mason University, Fairfax, VA, USA New Century College, George Mason University, Fairfax, VA, USA
a r t i c l e
i n f o
Article history: Received 20 March 2014 Accepted 7 April 2014 Available online xxxx Keywords: Maned wolf Osmotic stress Sperm cryopreservation Glycerol Dimethyl sulfoxide Cooling
a b s t r a c t We assessed the influences of medium osmolality, cryoprotectant and cooling and warming rate on maned wolf (Chrysocyon brachyurus) spermatozoa. Ejaculates were exposed to Ham’s F10 medium (isotonic control) or to this medium plus NaCl (350–1000 mOsm), sucrose (369 and 479 mOsm), 1 M glycerol (1086 mOsm) or dimethyl sulfoxide (Me2SO, 1151 mOsm) for 10 min. Each sample then was diluted back into Ham’s medium and assessed for sperm motility and plasma membrane integrity. Although glycerol and Me2SO had no influence (P > 0.05), NaCl and sucrose solutions affected sperm motility (P < 0.05), but not membrane integrity. Motility of sperm exposed to 0.05) to the control. As osmolality of the NaCl solution increased, motility decreased to 0.05) sperm motility metrics to the isotonic control, but lower (P < 0.05) than measured in raw ejaculate. Dilution in higher osmolalities of NaCl markedly reduced (P < 0.05; 600 mOsm) or completely eliminated (800, 1000 mOsm) motility. By contrast, a hypertonic solution of glycerol or Me2SO protected cellular motility, resulting in values no different (P > 0.05) from the isotonic control or raw ejaculate. However, the 0.1 or 0.2 M sucrose solution (360 versus 480 mOsm, respectively) was unable (P < 0.05) to sustain sperm motility at the raw ejaculate level, although there was no difference (P > 0.05) from the isotonic control. Study 2: Cryoprotectant, cooling and warming influences on cryosurvival of maned wolf spermatozoa Overall, across all treatments and regardless of cryopreservation procedure, freezing and thawing adversely (P < 0.05) influenced maned wolf spermatozoa compared to values for unfrozen, raw ejaculate (sperm motility, 16.8 ± 1.9% versus 72.5 ± 2.5%; acrosomal integrity, 54.0 ± 1.6% versus 76.1 ± 2.5; plasma membrane integrity, 58.3 ± 1.8% versus 82.4 ± 1.9%, respectively). From the LME models (Tables 4–6), cryoprotectant, time and interaction of cryoprotectant and cooling method most affected post-thaw sperm motility (bolded values; Table 4). Analyses revealed that samples cryopreserved in 1 M Me2SO exhibited superior (P = 0.03) motility (20.0 ± 1.9%) immediately post-thaw compared to those frozen in 1 M glycerol (13.5 ± 2.1%). Additionally, incubating spermatozoa at 38.5 °C for 1 h after diluting into isotonic medium reduced motility by about 50% (8.6 ± 0.9%) compared to that measured before dilution (16.7 ± 1.4%) and immediately post-dilution (15.5 ± 1.3%). The model demonstrated that only cooling method influenced cellular motility when glycerol was the CPA (Table 4). In this analysis, those samples cooled more slowly (top rack method) retained better motility (19.7 ± 2.4%) than those exposed to faster cooling (either on the bottom rack, 6.6 ± 1.2%, P < 0.0001; or using the dry shipper method, 10.1 ± 1.5%, P = 0.001). In the presence of Me2SO, cooling rate had no impact
(P > 0.05) on sperm motility (top rack, 22.7 ± 4.00; bottom rack, 19.6 ± 3.2; dry shipper, 17.9 ± 2.8). Thawing methods had no effect on sperm motility at any time for either CPA. There was no influence (P > 0.05) of cryoprotectant, cooling or warming method on acrosomal integrity (Table 5). However, time of incubation after thawing was an influential factor. The proportion of spermatozoa with intact acrosomes declined (P < 0.001) from 54.0 ± 1.7% immediately post-thawing to 22.8 ± 1.7% at 1 h of in vitro incubation. By contrast, both cryoprotectant and time influenced percentages of spermatozoa with intact plasma membranes (Table 6). The use of Me2SO improved (P < 0.0001) membrane integrity compared to glycerol (51.2 ± 1.7% versus 41.5 ± 2.2%, respectively). Overall, membrane integrity was adversely affected (P = 0.000) by the 1 h incubation (58.3 ± 2.4%, immediate post-thaw versus 33.8 ± 1.8%, 1 h later). Neither cooling nor warming method affected plasma membrane integrity.
Discussion Compared to substantial data available on wild felids [review[66]], the amount of specific information on basic reproductive physiology, including gamete sensitivity to cold temperature, is rudimentary for the Canidae family. There are 36 wild species of canids [28], all of which have been far less studied than felids, in part, due to fewer animals available in ex situ collections for hypothesis-based research. Species in this taxon are extreme in their reproductive diversity, with significant variations in social structure (individual versus pack configurations), time and frequency of mating activity (seasonal versus non-seasonal breeding) and even type of ovulatory mechanism (spontaneous versus induced) [65]. Particularly challenging has been the ability to recover high quality electroejaculate, especially from a species like the maned wolf that has such a narrow (8 wk long) window of active spermatogenesis and sperm production [8,55]. Clearly, based on earlier observations, [8,55] and our findings here, the quality of maned wolf semen recovered by electroejaculation is consistently less that that reported for the domestic dog where semen is routinely collected using an artificial vagina [45]. Numbers of total sperm per maned wolf electroejaculate also have been less and proportions of pleiomorphic cells more than reported for the Mexican gray wolf (Canis lupus baileyi, 756.2 106 total spermatozoa [68,69]), red wolf (C. rufus, 349.4 106 total spermatozoa [23]) or generic gray wolf (Canis lupus; 1597.4 106 total spermatozoa [69]) collected in a similar fashion. The values for the maned wolf indeed appear to be species ‘norms’ as similar metrics (low sperm density and elevated proportions of cellular malformations) have been recorded for conspecifics captured, anesthetized and electroejaculated in nature [55]. It has long been established that there is a relationship between low levels of genetic diversity and compromised sperm structure and function in carnivores [44], including the domestic dog [63] and Mexican gray wolf [2]. Maned wolves, both those in ex situ collections as well as living in the wild, have low heterozygosity (nucleotide diversity, p = 0.0013) due to a genetic bottleneck during the last glacial period [40]). Because our ultimate goal is to incorporate AI with frozenthawed spermatozoa into the ex situ breeding program of the maned wolf, our purpose here was to begin understanding the sensitivity of the spermatozoa to primary factors normally encountered during the freeze–thaw process. These included differing osmolalities, cryo-agents, cooling and warming rates and removal of the cryoprotectant (i.e., dilution). Although it was not surprising that the overall viability of maned wolf spermatozoa would decrease due to freeze–thawing, the magnitude of the decline was unexpected, with 63–93% of motility and 22–88% of plasma
Please cite this article in press as: A.E.M. Johnson et al., Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury, but respond favorably to dimethyl sulfoxide, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.04.004
7
A.E.M. Johnson et al. / Cryobiology xxx (2014) xxx–xxx
Fig. 4. Mean ± S.E.M. of cellular motility (a) and membrane integrity (b) of freshly-ejaculated maned wolf spermatozoa (n = 7 ejaculates) after exposure to varying osmolalities of a NaCl solution or permeating (glycerol versus Me2SO) or non-permeating (sucrose) cryoprotectant followed by dilution back into isotonic medium. Bars with different superscripts are different (P < 0.05).
Table 4 Results from Linear Mixed Effect model predicting the influence of cryoprotectant, cooling method, warming rate and time on post-thaw motility in the maned wolf.a Estimate ± SEM Full model Intercept 19.60 ± 2.38 Cryoprotectant (Me2SO) Glycerol 10.39 ± 2.80 Cooling method (bottom rack) Dry shipper 3.44 ± 2.28 Top rack 2.55 ± 2.33 Thawing method (37 °C) 50 °C 0.22 ± 1.32 Time (0 h post-dilution into isotonic medium) Before dilution 1.19 ± 1.62 1 h post-dilution 6.89 ± 1.62 Cryoprotectant (glycerol) cooling method (bottom rack) Glycerol dry shipper 6.94 ± 3.20 Glycerol top rack 15.67 ± 3.26
d.f.
t Value
P value
AIC & BIC
202
8.25
0.00
1562.48 1598.98
202
4.56
0.00
202 202
1.51 1.09
0.13 0.27
202
0.17
0.87
202 202
0.73 4.24
0.46 0.00
202 202
2.15 4.89
0.03 0.00
LME summary of each effect was reported as a t-test [70]. a Treatments listed are compared to treatments in parenthesis for each variable (cryoprotectants, cooling method, time and an interaction of cryoprotectant and cooling method). Mean values are ± S.E.M. Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) were used as a guide for model selection. Bold values indicate significant difference.
membrane integrity lost between immediate collection and post-thaw assessment. While specific causes for this damage require further study, we made four important observations. First,
spermatozoa from this wild canid were highly susceptible to exposure to a hypertonic salt (NaCl) solution. Second, the type of CPA was important, with clear advantages for Me2SO over glycerol in
Please cite this article in press as: A.E.M. Johnson et al., Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury, but respond favorably to dimethyl sulfoxide, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.04.004
8
A.E.M. Johnson et al. / Cryobiology xxx (2014) xxx–xxx
Table 5 Results from Linear Mixed Effect model predicting the effects of cryoprotectant, cooling method, warming rate and time on post-thaw acrosomal integrity in the maned wolf.* Estimate ± SEM Full model Intercept 51.81 ± 6.79 Cryoprotectant (Me2SO) Glycerol 2.44 ± 1.80 Cooling method (bottom rack) Dry shipper 0.75 ± 2.19 Top rack 3.05 ± 2.22 Thawing method (37 °C) 50 °C 3.10 ± 1.80 Time (0 h post-dilution into isotonic medium) Before dilution 25.09 ± 2.24 1 h post-dilution 4.65 ± 2.19
d.f.
t Value
P value
AIC & BIC 1422.73 1451.21
173
7.63
0.00
173
1.36
0.17
173 173
0.34 1.38
0.73 0.17
173
1.72
0.09
173 173
11.18 2.12
0.00 0.03
LME summary of each effect was reported as a t-test [70]. Treatments listed are compared to treatments in parenthesis for each variable (cryoprotectants, cooling method, time and an interaction of cryoprotectant and cooling method). Mean values are ± S.E.M. Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) were used as a guide for model selection. Bold values indicate significant difference.
*
Table 6 Results from a Linear Mixed Effect model predicting the influence of cryoprotectant, cooling method, warming rate and time on post-thaw sperm plasma membrane integrity in the maned wolf.* Estimate ± SEM Full model Intercept 57.00 ± 3.46 Cryoprotectant (Me2SO) Glycerol 9.41 ± 2.63 Cooling method (bottom rack) Dry shipper 3.49 ± 3.20 Top rack 5.46 ± 3.23 Thawing method (37 °C) 50 °C 4.74 ± 2.63 Time (0 h post-dilution into isotonic medium) Before dilution 11.37 ± 3.20 1 h post-dilution 13.60 ± 3.23
d.f.
t Value
P value
AIC & BIC
169
16.47
0.00
1509.67 1537.95
169
3.58
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury, but respond favorably to dimethyl sulfoxide q,qq Amy E.M. Johnson a,b, Elizabeth W. Freeman c, David E. Wildt a, Nucharin Songsasen a,⇑ a b c
Center for Species Survival, Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA, USA Department of Environmental Science and Policy, George Mason University, Fairfax, VA, USA New Century College, George Mason University, Fairfax, VA, USA
a r t i c l e
i n f o
Article history: Received 20 March 2014 Accepted 7 April 2014 Available online xxxx Keywords: Maned wolf Osmotic stress Sperm cryopreservation Glycerol Dimethyl sulfoxide Cooling
a b s t r a c t We assessed the influences of medium osmolality, cryoprotectant and cooling and warming rate on maned wolf (Chrysocyon brachyurus) spermatozoa. Ejaculates were exposed to Ham’s F10 medium (isotonic control) or to this medium plus NaCl (350–1000 mOsm), sucrose (369 and 479 mOsm), 1 M glycerol (1086 mOsm) or dimethyl sulfoxide (Me2SO, 1151 mOsm) for 10 min. Each sample then was diluted back into Ham’s medium and assessed for sperm motility and plasma membrane integrity. Although glycerol and Me2SO had no influence (P > 0.05), NaCl and sucrose solutions affected sperm motility (P < 0.05), but not membrane integrity. Motility of sperm exposed to 0.05) to the control. As osmolality of the NaCl solution increased, motility decreased to 0.05) sperm motility metrics to the isotonic control, but lower (P < 0.05) than measured in raw ejaculate. Dilution in higher osmolalities of NaCl markedly reduced (P < 0.05; 600 mOsm) or completely eliminated (800, 1000 mOsm) motility. By contrast, a hypertonic solution of glycerol or Me2SO protected cellular motility, resulting in values no different (P > 0.05) from the isotonic control or raw ejaculate. However, the 0.1 or 0.2 M sucrose solution (360 versus 480 mOsm, respectively) was unable (P < 0.05) to sustain sperm motility at the raw ejaculate level, although there was no difference (P > 0.05) from the isotonic control. Study 2: Cryoprotectant, cooling and warming influences on cryosurvival of maned wolf spermatozoa Overall, across all treatments and regardless of cryopreservation procedure, freezing and thawing adversely (P < 0.05) influenced maned wolf spermatozoa compared to values for unfrozen, raw ejaculate (sperm motility, 16.8 ± 1.9% versus 72.5 ± 2.5%; acrosomal integrity, 54.0 ± 1.6% versus 76.1 ± 2.5; plasma membrane integrity, 58.3 ± 1.8% versus 82.4 ± 1.9%, respectively). From the LME models (Tables 4–6), cryoprotectant, time and interaction of cryoprotectant and cooling method most affected post-thaw sperm motility (bolded values; Table 4). Analyses revealed that samples cryopreserved in 1 M Me2SO exhibited superior (P = 0.03) motility (20.0 ± 1.9%) immediately post-thaw compared to those frozen in 1 M glycerol (13.5 ± 2.1%). Additionally, incubating spermatozoa at 38.5 °C for 1 h after diluting into isotonic medium reduced motility by about 50% (8.6 ± 0.9%) compared to that measured before dilution (16.7 ± 1.4%) and immediately post-dilution (15.5 ± 1.3%). The model demonstrated that only cooling method influenced cellular motility when glycerol was the CPA (Table 4). In this analysis, those samples cooled more slowly (top rack method) retained better motility (19.7 ± 2.4%) than those exposed to faster cooling (either on the bottom rack, 6.6 ± 1.2%, P < 0.0001; or using the dry shipper method, 10.1 ± 1.5%, P = 0.001). In the presence of Me2SO, cooling rate had no impact
(P > 0.05) on sperm motility (top rack, 22.7 ± 4.00; bottom rack, 19.6 ± 3.2; dry shipper, 17.9 ± 2.8). Thawing methods had no effect on sperm motility at any time for either CPA. There was no influence (P > 0.05) of cryoprotectant, cooling or warming method on acrosomal integrity (Table 5). However, time of incubation after thawing was an influential factor. The proportion of spermatozoa with intact acrosomes declined (P < 0.001) from 54.0 ± 1.7% immediately post-thawing to 22.8 ± 1.7% at 1 h of in vitro incubation. By contrast, both cryoprotectant and time influenced percentages of spermatozoa with intact plasma membranes (Table 6). The use of Me2SO improved (P < 0.0001) membrane integrity compared to glycerol (51.2 ± 1.7% versus 41.5 ± 2.2%, respectively). Overall, membrane integrity was adversely affected (P = 0.000) by the 1 h incubation (58.3 ± 2.4%, immediate post-thaw versus 33.8 ± 1.8%, 1 h later). Neither cooling nor warming method affected plasma membrane integrity.
Discussion Compared to substantial data available on wild felids [review[66]], the amount of specific information on basic reproductive physiology, including gamete sensitivity to cold temperature, is rudimentary for the Canidae family. There are 36 wild species of canids [28], all of which have been far less studied than felids, in part, due to fewer animals available in ex situ collections for hypothesis-based research. Species in this taxon are extreme in their reproductive diversity, with significant variations in social structure (individual versus pack configurations), time and frequency of mating activity (seasonal versus non-seasonal breeding) and even type of ovulatory mechanism (spontaneous versus induced) [65]. Particularly challenging has been the ability to recover high quality electroejaculate, especially from a species like the maned wolf that has such a narrow (8 wk long) window of active spermatogenesis and sperm production [8,55]. Clearly, based on earlier observations, [8,55] and our findings here, the quality of maned wolf semen recovered by electroejaculation is consistently less that that reported for the domestic dog where semen is routinely collected using an artificial vagina [45]. Numbers of total sperm per maned wolf electroejaculate also have been less and proportions of pleiomorphic cells more than reported for the Mexican gray wolf (Canis lupus baileyi, 756.2 106 total spermatozoa [68,69]), red wolf (C. rufus, 349.4 106 total spermatozoa [23]) or generic gray wolf (Canis lupus; 1597.4 106 total spermatozoa [69]) collected in a similar fashion. The values for the maned wolf indeed appear to be species ‘norms’ as similar metrics (low sperm density and elevated proportions of cellular malformations) have been recorded for conspecifics captured, anesthetized and electroejaculated in nature [55]. It has long been established that there is a relationship between low levels of genetic diversity and compromised sperm structure and function in carnivores [44], including the domestic dog [63] and Mexican gray wolf [2]. Maned wolves, both those in ex situ collections as well as living in the wild, have low heterozygosity (nucleotide diversity, p = 0.0013) due to a genetic bottleneck during the last glacial period [40]). Because our ultimate goal is to incorporate AI with frozenthawed spermatozoa into the ex situ breeding program of the maned wolf, our purpose here was to begin understanding the sensitivity of the spermatozoa to primary factors normally encountered during the freeze–thaw process. These included differing osmolalities, cryo-agents, cooling and warming rates and removal of the cryoprotectant (i.e., dilution). Although it was not surprising that the overall viability of maned wolf spermatozoa would decrease due to freeze–thawing, the magnitude of the decline was unexpected, with 63–93% of motility and 22–88% of plasma
Please cite this article in press as: A.E.M. Johnson et al., Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury, but respond favorably to dimethyl sulfoxide, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.04.004
7
A.E.M. Johnson et al. / Cryobiology xxx (2014) xxx–xxx
Fig. 4. Mean ± S.E.M. of cellular motility (a) and membrane integrity (b) of freshly-ejaculated maned wolf spermatozoa (n = 7 ejaculates) after exposure to varying osmolalities of a NaCl solution or permeating (glycerol versus Me2SO) or non-permeating (sucrose) cryoprotectant followed by dilution back into isotonic medium. Bars with different superscripts are different (P < 0.05).
Table 4 Results from Linear Mixed Effect model predicting the influence of cryoprotectant, cooling method, warming rate and time on post-thaw motility in the maned wolf.a Estimate ± SEM Full model Intercept 19.60 ± 2.38 Cryoprotectant (Me2SO) Glycerol 10.39 ± 2.80 Cooling method (bottom rack) Dry shipper 3.44 ± 2.28 Top rack 2.55 ± 2.33 Thawing method (37 °C) 50 °C 0.22 ± 1.32 Time (0 h post-dilution into isotonic medium) Before dilution 1.19 ± 1.62 1 h post-dilution 6.89 ± 1.62 Cryoprotectant (glycerol) cooling method (bottom rack) Glycerol dry shipper 6.94 ± 3.20 Glycerol top rack 15.67 ± 3.26
d.f.
t Value
P value
AIC & BIC
202
8.25
0.00
1562.48 1598.98
202
4.56
0.00
202 202
1.51 1.09
0.13 0.27
202
0.17
0.87
202 202
0.73 4.24
0.46 0.00
202 202
2.15 4.89
0.03 0.00
LME summary of each effect was reported as a t-test [70]. a Treatments listed are compared to treatments in parenthesis for each variable (cryoprotectants, cooling method, time and an interaction of cryoprotectant and cooling method). Mean values are ± S.E.M. Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) were used as a guide for model selection. Bold values indicate significant difference.
membrane integrity lost between immediate collection and post-thaw assessment. While specific causes for this damage require further study, we made four important observations. First,
spermatozoa from this wild canid were highly susceptible to exposure to a hypertonic salt (NaCl) solution. Second, the type of CPA was important, with clear advantages for Me2SO over glycerol in
Please cite this article in press as: A.E.M. Johnson et al., Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury, but respond favorably to dimethyl sulfoxide, Cryobiology (2014), http://dx.doi.org/10.1016/j.cryobiol.2014.04.004
8
A.E.M. Johnson et al. / Cryobiology xxx (2014) xxx–xxx
Table 5 Results from Linear Mixed Effect model predicting the effects of cryoprotectant, cooling method, warming rate and time on post-thaw acrosomal integrity in the maned wolf.* Estimate ± SEM Full model Intercept 51.81 ± 6.79 Cryoprotectant (Me2SO) Glycerol 2.44 ± 1.80 Cooling method (bottom rack) Dry shipper 0.75 ± 2.19 Top rack 3.05 ± 2.22 Thawing method (37 °C) 50 °C 3.10 ± 1.80 Time (0 h post-dilution into isotonic medium) Before dilution 25.09 ± 2.24 1 h post-dilution 4.65 ± 2.19
d.f.
t Value
P value
AIC & BIC 1422.73 1451.21
173
7.63
0.00
173
1.36
0.17
173 173
0.34 1.38
0.73 0.17
173
1.72
0.09
173 173
11.18 2.12
0.00 0.03
LME summary of each effect was reported as a t-test [70]. Treatments listed are compared to treatments in parenthesis for each variable (cryoprotectants, cooling method, time and an interaction of cryoprotectant and cooling method). Mean values are ± S.E.M. Akaike Information Criterion (AIC) and Bayesian Information Criterion (BIC) were used as a guide for model selection. Bold values indicate significant difference.
*
Table 6 Results from a Linear Mixed Effect model predicting the influence of cryoprotectant, cooling method, warming rate and time on post-thaw sperm plasma membrane integrity in the maned wolf.* Estimate ± SEM Full model Intercept 57.00 ± 3.46 Cryoprotectant (Me2SO) Glycerol 9.41 ± 2.63 Cooling method (bottom rack) Dry shipper 3.49 ± 3.20 Top rack 5.46 ± 3.23 Thawing method (37 °C) 50 °C 4.74 ± 2.63 Time (0 h post-dilution into isotonic medium) Before dilution 11.37 ± 3.20 1 h post-dilution 13.60 ± 3.23
d.f.
t Value
P value
AIC & BIC
169
16.47
0.00
1509.67 1537.95
169
3.58
