Introduction
Semen cryopreservation is an efficient method to extend genetic materials among a population especially those on the brink of extinction. Furthermore, the success of breeding programmes namely artificial insemination (AI) relies mostly on the quality of fresh or cryopreserved semen as in the case with camelid species. It is speculated that Bactrian camel (Camelus bactrianus) was domesticated on the eastern border of the Caspian Sea in 2500 BC (Fowler, Reference Fowler1997). However, its population is on the edge of extinction in Iran, with less than 156 camels (Niasari-Naslaji et al., Reference Niasari-Naslaji, Nikjou, Skidmore, Moghiseh, Mostafaey, Razavi and Moosavi-Movahedi2009). Although the initial success report on AI in Bactrian camel dates back to early 1960s (Elliot, Reference Elliot1961), limited numbers of studies have reported pregnancies from AI either by fresh or frozen–thawed semen (Zhao et al., Reference Zhao, Huang and Chen1994; Bravo et al., Reference Bravo, Skidmore and Zhao2000). Some biochemical and biophysical traits of camelid semen have been characterized although its manipulation and cryopreservation is still a tricky process because of poor quality and highly viscous ejaculates (Bravo et al., Reference Bravo, Skidmore and Zhao2000). This situation, in turn, ends in ineffective cryopreservation, therefore hindering the extensive application of AI in this family.
Previous reports have suggested that the diluent, cryoprotectant type and cryoprotectant exposure time, as well as freezing and thawing rates, are important criteria to develop suitable and species-specific cryopreservation protocols (Morton et al., Reference Morton, Evans and Maxwell2010; Ahmad et al., Reference Ahmad, Nasrullah and Ahmad2015). As for freezing extender, introducing SHOTOR diluent in Bactrian camel (Niasari-Naslaji et al., Reference Niasari-Naslaji, Mosaferi, Bahmani, Gerami, Gharahdaghi, Abarghani and Ghanbari2007) and Tris–citrate–fructose buffer in Dromedary camel (Malo et al., Reference Malo, Crichton and Skidmore2017) resulted in satisfactory sperm quality after freezing–thawing.
The freezability of Bactrian camel spermatozoa in different dilution rates (DR) has not been assessed.
In other words, being an important determinant of cryopreservation outcome, Camelid sperm DR has been mostly studied in liquid storage with contradictory results. For instance, motility and acrosome integrity of ejaculated alpaca sperm with different DRs (1:1, 1:2 or 1:4; v:v) stored at 5 or 15ºC for different storage time (0, 24, 48 or 72 h) showed the highest value of four-fold diluted samples in all temperatures and storage times (Morton et al., Reference Morton, Gibb, Bertoldo and Maxwell2009). Conversely, 1:3 and 1:10 DRs were utilized for Dromedary and Bactrian sperm storage, respectively (Deen et al., Reference Deen, Vyas, Jain and Sahani2004; Niasari-Naslaji et al., Reference Niasari-Naslaji, Mosaferi, Bahmani, Gharahdaghi, Abarghani, Ghanbari and Gerami2006).
The equilibration time (ET) is another contributing factor defined as a pre-freezing period during which glycerol quickly penetrates into the spermatozoon to establish equilibrium between its intracellular and extracellular compartments (Shah et al., Reference Shah, Andrabi and Qureshi2016). Although long exposure to glycerol seems to be detrimental for spermatozoa, an optimum period is required for the plasma membrane to adopt to lower temperatures. Water outflow through cells resulted in lower damage caused by ice crystals during freezing–thawing (Fleisch et al., Reference Fleisch, Malama, Witschi, Leiding, Siuda, Janett and Bollwein2017). A broad range of equilibration periods from no equilibration at all to 3 h has been reported in different species (Leite et al., Reference Leite, Do Vale Filho, De Arruda, De Andrade, Emerick, Zaffalon, Martins and De Andrade2010; Kaka et al. Reference Kaka, Wahid, Rosnina, Yimer, Khumran, Sarsaifi, Behan, Kaka and Ebrahimi2015; Pradiee et al., Reference Pradiee, O’Brien, Esteso, Castaño, Toledano-Díaz, López-Sebastián, Marcos-Beltran, Vega, Guillamon, Martinez-Nevado, Guerra and Santiago-Moreno2016). A greater range (0–4 h) with no direct comparison and with contradictory results has also been reported in camelid semen cryopreservation (Niasari-Naslaji et al., Reference Niasari-Naslaji, Mosaferi, Bahmani, Gerami, Gharahdaghi, Abarghani and Ghanbari2007; Abd El-Salaam Reference Abd El-Salaam2013; Malo et al., Reference Malo, Crichton and Skidmore2017).
Generally, studies on the Bactrian camel spermatozoa cryopreservation are scant in the literature and, indeed, a broad range of 25–70 × 106 sperm cell/straw was applied with no direct comparison (Niasari-Naslaji et al., Reference Niasari-Naslaji, Mosaferi, Bahmani, Gerami, Gharahdaghi, Abarghani and Ghanbari2007). Additional research is warranted to develop a feasible strategy to cryopreserve Bactrian camel spermatozoa. Therefore, this study was conducted to determine an optimum DR and ET for its semen freezing ability.
Materials and methods
Ethics
The local Ethic Committee of Mohaghegh Ardbili University (Ardabil, Iran) approved issues concerning the experimental setups and evaluation techniques.
Chemicals
All chemicals were purchased from the Sigma-Aldrich Co. (St. Louis, MO, USA) and Merck (Darmstadt, Germany).
Animals
Four healthy adult male camels, aged 6–12 years old, were housed individually at Bactrian Camel Research Center, Jahadabad, Meshkinshahr, Ardabil Province, Iran. Animals were exposed to natural day length with ambient temperature and fed a diet containing 7.5 kg alfalfa hay and 2.5 kg mixed concentrate including 68% barley, 12% cotton seed meal, 17% wheat bran, 2% molasses, and 1% mixed vitamins and minerals.
Semen collection, processing and cryopreservation
Semen sampling was carried out weekly during the breeding season via a modified bovine artificial vagina. Fresh semen samples were immediately transported to the laboratory and kept in a water bath at 37°C. Samples of good quality (>60% total motility, >80% viability) were pooled and subjected to a two-step freezing procedure. Different DRs achieved after extending pooled sample with various amount of pre-warmed SHOTOR extender without cryoprotectant (2× 1:1, 1:2, 1:4 and 1:8 ratios) and cooled to 4°C in 2 h. After inclusion of an appropriate amount of cryoprotectant (glycerol, a final concentration of 5%) samples were subjected to two ETs (1 or 2 h). They were then loaded in 0.25 ml straws (IMV, L’Aigle, France), sealed with polyvinyl alcohol powder (IMV, L’Aigle, France) and placed horizontally 4 cm above liquid nitrogen in a cryobox for 8 min. Finally, straws were plunged into liquid nitrogen and stored until evaluation. The thawing procedure was performed in a water bath at 37°C for 30 s. In total, eight straws were considered for each treatment and this experiment was repeated eight times. SHOTOR diluent composed of 214.6 mM Tris, 64.2 mM citric acid, 66.6 mM glucose, and 49.9 mM fructose with an osmolarity of 330 mOsm/kg (Niasari-Naslaji et al., Reference Niasari-Naslaji, Mosaferi, Bahmani, Gerami, Gharahdaghi, Abarghani and Ghanbari2007).
Gross assessment
Computer-assisted semen analysis parameters
For analyzing motility parameters, sperm samples were incubated after thawing in a water bath at 37°C for 5 min. A 5 μl aliquot of semen was placed directly on a pre-warmed microscope slide (76.2 × 24.5 mm; Pearl, China), covered by a coverslip (24 mm × 24 mm; Menzel-Glaser, Germany) and sperm motility characteristics were determined using a phase-contrast microscope (Nikon, Japan) equipped with computer-assisted semen analysis (CASA) software (Hoshmand Fanavaran, Version 6, Amirkabir Medical Engineering Co., Tehran, Iran). The assessed parameters included total motility (TM, %), progressive motility (PM, %), straight line velocity (VSL, µm/s), curvilinear velocity (VCL, µm/s), average path velocity (VAP, %), straightness (STR, %), linearity (%), amplitude of lateral head displacement (ALH, µm), and beat-cross frequency (BCF, Hz). CASA parameters for the camel species were adapted from Malo et al. (Reference Malo, Elwing, Soederstroem, Lundeheim, Morrell and Skidmore2019). The system parameters for CASA were a frame rate of 30/s, Particles of size 13–101 µm, progressive STR (%) 70, progressive VAP (µm/s) 40, slow VAP (µm/s) 20, slow VSL (µm/s) 30, static VAP (µm/s) 4, and static VSL (µm/s) 1. For each evaluation, 10 fields were assessed to include at least 200 spermatozoa.
Viability
Sperm viability was assessed using the eosin–nigrosin staining method (eosin Y 0.67 g, nigrosin 10 g, NaCl 0.9 g dissolved in 100 ml double-distilled water). Briefly,10 µl of diluted semen was mixed with 20 µl of stain on a pre-warmed slide and smeared. Each slide was prepared in duplicate and the percentages of unstained heads of spermatozoa (live) and stained/partial stained heads of spermatozoa (dead) were determined by counting 200 spermatozoa under a phase-contrast microscope at ×400 magnification (Seifi-Jamadi et al., Reference Seifi-Jamadi, Kohram, Shahneh, Ansari and Macías-García2016).
Lipid peroxidation
Lipid peroxidation is simply the oxidative degradation of mainly plasma membrane lipids from which free radicals grab electrons and subsequently results in cell damage. Malondialdehyde is the most prevalent byproduct of lipid peroxidation, so that its measurement by thiobarbituric acid assay is widely acknowledged as the technique to assess oxidative damage (Esterbauer and Cheeseman, Reference Esterbauer and Cheeseman1990). Here, 1 ml of diluted semen (1:2 in 2.9% sodium citrate solution) was first mixed with 2 ml of trichloroacetic acid (TCA; 612 mM), 1 ml of ethylenediaminetetraacetic acid (EDTA; 12.66 mM) and 1ml of butylated hydroxytoluene (BHT; 90.76 mM in ethyl alcohol 100%). After centrifugation (900 g, 18°C for 10 min), 1 ml of supernatant was incubated with 1 ml of thiobarbituric acid (TBA; 3.6 mM in acetic acid solution; pH: 4) in a boiling water bath for 10 min. After cooling, the absorbance at 532 nm was read using a spectrophotometer.
Hypo-osmotic swilling test
The hypo-osmotic swelling (HOS) test was used to determine spermatozoa with intact plasma membranes. Briefly, 10 µl of diluted semen was mixed with 100 µl of a 100 mOsm hypo-osmotic solution (9 g fructose + 4.9 g sodium citrate per litre of bi-distilled water) in a 0.5-ml test tube. After 45 min incubation at room temperature, the mixtures were homogenized. Then, a drop of fixed semen was placed on a warm slide and spread with a coverslip. The percentage of spermatozoa with a curled or swollen flagellum was estimated by counting 200 sperm per slide using a phase-contrast microscope at ×400 magnification (Niasari-Naslaji et al., Reference Niasari-Naslaji, Mosaferi, Bahmani, Gerami, Gharahdaghi, Abarghani and Ghanbari2007).
Sperm morphology
The Schafer and Holzmann (Reference Schafer and Holzmann2000) method was applied in Hancock’s solution to evaluate sperm morphology. Briefly, sodium saline solution (9.01 g NaCl/500 ml double-distilled water) and buffer solutions (A, 21.682 g Na2HPO4/500 ml double-distilled water; B, 22.254 g KH2PO4/500 ml double-distilled water). Then 200 ml buffer A and 80 ml buffer B was mixed to prepare 280 ml of buffer solution C. The final Hancock solution was composed of 62.5 ml formalin (37%) + 150 ml sodium saline solution + 150 ml buffer solution C, and 500 ml double-distilled water. A 5-µl aliquot of diluted semen was mixed with 500 µl Hancock’s solution. A sample of this mixture was deposited on a slide, covered with a cover slide and sperm abnormality percentage was estimated by counting those with detached heads, abaxial heads, malformed heads, bent tails, coiled tails, double tails and protoplasmic droplets in 200 spermatozoa per slide using a phase-contrast microscope at ×400 magnification
Statistical analysis
Data were tested for normality using the univariate procedure and the Shapiro–Wilk test. Arc sin transformation (Arc sin) was performed when necessary. Statistical analysis of measurement data was performed using PROC GLM of SAS version 9 (SAS Institute Inc., Cary, NC, USA). The statistical model was as follows: Yij = µ + Ti + eij, where Yijk is the parameter evaluated, µ is the mean of the population, Ti is the mean of treatments and eijk is residual effect. Tukey’s test was applied to compare lsmeans and a cut-off of 5% was used to indicate statistical significance. Results were presented as the Lsmean ± standard error (SE).
Results
Objective parameters
Post-thaw sperm kinematic parameters in two ETs and at four DRs are presented in Table 1. Overall, it can be seen that, despite some fluctuations, a four-fold dilution along with 1 h of ET recorded more satisfying results compared with other treatments (P < 0.05).
Effect of different dilution rates and equilibration times on post-thaw quality of Bactrian camel spermatozoa assessed by CASA
Lsmeans with different superscripts (A–B, a–b) in a column are significantly (P < 0.05) different. The assessed parameters included motility (TM, %), progressive motility (PM, %), straight line velocity (VSL, µm/s), curvilinear velocity (VCL, µm/s), average path velocity (VAP, %), straightness (STR, %), and linearity (%), amplitude of lateral head displacement (ALH, µm), and beat-cross frequency (BCF, Hz).
Moving to a more detailed analysis, sperm total motility percentage was significantly higher in samples with four-fold dilution (39.58%) than in both the lowest (31.83%) and highest (33.33%) diluted samples (Table 1). Similarly, a 1:4 DR could significantly enhance sperm PM percentage compared with other DRs (19.50 vs 14.00, 16:00 and 14.25 for 1:4, 1:1, 1:2 and 1:8 DR, respectively).
For ET, increasing the ET from 1 to 2 h adversely affected sperm total motility after freezing–thawing. A 1 h ET prior to cryopreservation resulted in an ~4% increase in sperm total motility after thawing, compared with 2 h of ET (P < 0.05). Furthermore, linearity (42.29 vs 32.26%), ALH (1.22 vs 0.86%) and BCF (13.15 vs 8.70%) percentages were significantly higher in 1 h of ET than in 2 h. Conversely, a significant enhancement in VCL (~15%) was recorded in the samples belonging to 2 h of ET (Table 1).
Subjective parameters
Samples in the 1:2 and 1:4 dilution groups had significantly higher live spermatozoa than the 1:1 group (Table 2; 51.75 and 55.37 vs 43.50%, respectively). Similarly, the highest significant value (P < 0.01) of membrane integrity was noted only at a 1:4 ratio compared with other ratios (46.75 vs 37.25, 42.62 and 37.37% for 1:4, 1:1, 1:2 and 1:8 DRs, respectively).
Effect of various dilution rates and equilibration times on some parameters of Bactrian camel spermatozoa after freezing–thawing
Lsmeans with different superscripts (A–B, a–c) in a column are significantly (P < 0.05) different.
For ETs, samples that underwent 2 h of ET compared with 1 h of ET, experienced a significant decline in sperm viability (51.37 vs 48.12%) and plasma membrane integrity percentages (42.25 vs 39.75%) (Table 2; P < 0.05). Although not statistically significant (P = 0.12), peroxidation increased following a longer ET (4.96 vs 5.66 for 1 and 2 h of ET, respectively).
Discussion
This study examined the effect of varying DRs and ETs on the post-thaw quality of Bactrian camel spermatozoa. A four-fold DR resulted in more satisfying results in terms of sperm motion parameters, viability and plasma membrane integrity. However, a lower dilution of spermatozoa before freezing led to poor post-thaw quality parameters. Although it has been proposed that increasing semen concentration, especially for animals with lower freezability of spermatozoa, can compensate quality loss during cryopreservation (Alvarez et al., Reference Alvarez, Tamayo-Canul, Anel, Boixo, Mata-Campuzano, Martinez-Pastor, Anel and de Paz2012), this, in turn, resulted in increased free radical production, modification of sperm metabolism, change in medium due to the catabolism products, and release of toxic products from damaged spermatozoa, all of which can contribute to the destabilization of membranes and other structures in intact spermatozoa (Chatterjee and Gagnon, Reference Chatterjee and Gagnon2001; Perez-Crespo et al., Reference Perez-Crespo, Moreira, Pintado and Gutierrez-Adan2008).
A considerable reduction in sperm motility, viability and membrane integrity was noted following cryopreservation at the higher DR (1:8). Negative effects of higher DR with sperm motility have been well documented for fowl (Austin and Natarajan, Reference Austin and Natarajan1991), boar (Bamba and Cran, 1988), and bull (Garner et al., Reference Garner, Thomas, Gravance, Marshall, DeJarnette and Allen2001) spermatozoa. This situation is believed to be caused by the interaction of seminal plasma with the diluent (Blesbois and De Reviers, Reference Blesbois and De Reviers1992). In fact, the proportion of cryoprotectant per sperm cell might regulate its function during cryopreservation (Daskin et al., Reference Daskin, Kulaksiz, Ergun and Halil2011). An optimum ratio may help sperm to survive ice crystal formation during freezing and thawing. Conversely, a higher DR might cause more glycerol to penetrate sperm cells, as a higher glycerol ratio per sperm cell is available, and consequently sperm quality would be diminished. Higher DRs may also reduce sperm post-thaw quality through dilution of several proteins or antioxidants present in seminal plasma that protect sperm against freezing–thawing (Viveiros and Leal, Reference Viveiros and Leal2016).
Regarding ET, 1 h compared with 2 h ET resulted in significantly higher sperm motility, LIN, ALH and BCF percentages in the present study. Given that cryoprotectant exposure time plays a pivotal role in cryopreservation success, it is important to determine the most appropriate time for each species. Although no comparison was made, Niasari-Naslaji et al. (Reference Niasari-Naslaji, Mosaferi, Bahmani, Gerami, Gharahdaghi, Abarghani and Ghanbari2007) applied an ET of 1 h for Bactrian camel sperm cryopreservation. In Dromedary camel, studies on the length of ET are contradictory. Considering a broad range of ET (10 min to 2 h), for instance, Malo et al. (Reference Malo, Crichton and Skidmore2017) reported no significant difference in post-thaw motility parameters. In another study, however, samples with no ET compared with 2 h or 4 h ET had significantly higher post-thaw motility and acrosome integrity (Abd El-Salaam, Reference Abd El-Salaam2013). In rooster, extremely short ET (no more than 1 min) resulted in lower viability of post-thaw sperm (Santiago-Moreno et al., Reference Santiago-Moreno, Castaño, Toledano-Díaz, Coloma, López-Sebastián, Prieto and Campo2011). Similarly, higher acrosome and plasma membrane damage, as well as motility loss, were noted in bull sperm given no ET (Leite et al., Reference Leite, Do Vale Filho, De Arruda, De Andrade, Emerick, Zaffalon, Martins and De Andrade2010). In the current study, samples subjected to 2 h of ET had lower plasma membrane integrity and viability compared with 1 h of ET. This finding might be the result of prolonged exposure to glycerol. The toxicity of cryoprotectant on sperm vital reactions such as glycolysis and fructose diphosphates has been well documented (Fahy et al., Reference Fahy, Lilley, Linsdell, Douglas and Meryman1990). In addition, the process of cryopreservation imposes various stresses not only on physical features of sperm, but also on sensitive organelles such as mitochondria that are required to produce ATP (Long, Reference Long2006). It is also worth mentioning that mitochondrial ATP is required for the maintenance of sperm motility and membrane integrity (Davila et al., Reference Davila, Munoz, Bolanos, Stout, Gadella, Tapia, da Silva, Ferrusola and Pena2016). Consequently, spermatozoa experience arduous conditions in terms of cryoprotectant toxicity and cryoinjuries during cryopreservation. Although the present study fails to disclose the plausible scenario behind sperm quality loss and higher cryoprotectant exposure time, all or at least some of the above-mentioned mechanisms might be involved.
In conclusion, the highest improvement in kinematic parameters, viability and membrane integrity of post-thawed sperm was obtained with a 1 h ET and 1:4 DR using the SHOTOR diluent. Therefore, this procedure may become an appropriate approach for cryopreservation of Bactrian camel sperm.
Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Conflicts of interest
None.
Ethical standards
The local Ethic Committee of Mohaghegh Ardbili University (Ardabil, Iran) approved issues concerning the experimental setups and evaluation techniques.
