Cryopreservation and its effects on motility and gene expression patterns and fertilizing potential of bovine epididymal sperm

Abstract Despite encountering new challenges in using epididymal sperm recovered from cauda epididymides, this accessible and, in some species, worthwhile sample makes inevitable the further development of a suitable cryopreservation protocol. In this study, sperm was recovered from the epididymis of 4°C overnight stored slaughtered bulls' testes and the effects of cryopreservation on the bovine epididymal sperm motility (with CASA) and gene expression patterns (with quantitative Real time‐PCR) were evaluated. Moreover the fertilizing potential of cryopreserved epididymal sperm was used in in vitro fertilization (IVF). After freezing and thawing of epididymal sperm, total and slow progressive sperm motility, VCL, VAP, MAD, ALH and BCF were significantly decreased (p < .05), while in the parameters of fast progressive motility, VSL, LIN, WOB and STR there were not any significant variations in the frozen sperm compared to fresh (non‐frozen) counterpart. The assessment of abundance of transcripts encoding motility (TSSK6) and fertility (PRM1 and PRM2)‐related genes in epididymal sperm, showed that these transcripts were affected by freezing especially in slow progressive motility status (p < .01). Furthermore, cleavage and blastocyst rate did not present any significant differences between bovine embryos produced in vitro by fresh or frozen‐thawed epididymal sperm. It can be concluded that epididymal sperm has enough freezability after overnight testes storage, and cryopreservation could not affect the percentage of in vitro produced embryos in spite of the changes of relative abundance of some transcripts and direction progressive motility pattern of sperm.


| INTRODUC TI ON
The use of epididymal sperm as a valuable source of male gamete for assisted reproductive techniques (ART) has recently escalated.
Sometimes, animal ART centres have easier and further access to this potential source in their researches and preserve the genetic ability of worth slaughtered animals or animals in the risk of extinction. Moreover this convenient source provides the conditions to do reproductive technologies such as artificial insemination (AI), IVF and so on. Also, applying the sperm cryopreservation technique allows a more economical and efficient way to use that material because it can be utilized anytime not just immediately after the death of an animal.
Some exclusive challenges arise when working with epididymal sperm. First, the majority of research has focused on increasing the effectiveness of cryopreservation and ART only utilizing ejaculated sperm. Second, epididymal and ejaculated sperm are different with regards to their exposure to seminal plasma. Epididymal sperm has been shown to have different respiration and metabolism rates, motility characteristics and plasma membrane integrity (Goovaerts et al., 2006;Miller, Winer, & Ax, 1990). The cauda epididymal sperm is less motile than ejaculated sperm and has also a lower velocity, straightness and linearity (Goovaerts et al., 2006). The epididymis of bull is very accessible and utilization of bovine as an animal model is expected to optimize the protocols of epididymal sperm cryopreservation for other species, specially human and endangered species.
Commonly, semen quality analysis has been considered as an initial option for fertility potential assessment. Visual parameters may not be adequate for a thorough evaluation of the fertility potential of semen. Several in vitro semen analysis methods have been proposed. Many laboratory techniques have been shown to confirm the fertility potential of a semen sample (Rodriguez-Martinez, 2003Rodriguez-Martinez & Barth, 2007). Laboratory-evaluated parameters of sperm quality include hyperactivated motility (Davis, Niswander, & Katz, 1992;Mortimer, 1990), sperm-oocyte interaction (Madrid-Bury et al., 2005;Rodriguez-Martinez, 2006) in addition to assessment of the integrity of various structures such as genomic DNA (gDNA), cell membrane, acrosome and mitochondria. In bovine cauda epididymal sperm cryopreservation, there are limited studies to the assessment of motility pattern of epididymal sperm with computer-assisted sperm analyzer (CASA) and gene expression fluctuations of some important regulatory transcripts involved in motility and fertility and their relation to egg fertilizing potential of these type of sperm subsequent of cryopreservation.
Based on the finding of some studies (Selvaraju, Krishnan, Archana, & Ravindra, 2016;Selvaraju et al., 2018;Shilpa et al., 2017), occurred changes in transcript levels of some genes during cryopreservation need to be well-defined to optimize cryopreservation and fertility procedures.
The aim of the present study was, therefore, evaluation of the effect of cryopreservation of epididymal sperm on the sperm motility and expression patterns of few selected genes related to motility (TSSK6) and fertility (PRM1 and PRM2) in epididymal sperm, and finally, find a relation between motility and gene expression patterns of evaluated genes and fertilizing potential in in vitro embryo production.

| Chemicals
Unless stated otherwise, chemicals were obtained from Sigma Chemical Co.

| Ethics statement
Animal husbandry and handling were conducted by the guidelines of the ethical committee of Shahrekord University, Iran.

| Sperm collection and motility assessment
To perform this study, randomly one testes of slaughtered crossbred bulls (n = 10), was removed and transferred to the laboratory within 2h at room temperature (19°C-22°C) and placed at 4°C overnight. In the lab, epididymides of testes were exposed by cutting the tunica vaginalis. Cauda epididymides were held with forceps, and multiple incisions were made in the tubuli with a bistoury. Sperm was collected with a blade and transferred to the Tris base medium (Tris: 200 mM, Citric acid: 66.7 mM, D-Fructose: 55.5 mM) and kept at room temperature for 1 hr to receive the maximum motility.
The sperm motility was determined by computer-assisted sperm analyzer (CASA; Hooshmand Fanavar) and those samples that had more than 50% motility, were used for experiments. Ten microlitre of sperm suspension was loaded on a pre-warmed Spermmeter semen analysis chamber (Sperm Processor Pvt. Ltd.; 10 µm depth) and then

| Cryopreservation and thawing of sperm
The cauda epididymal sperm cells from each bull were diluted slowly to a final concentration of 50 × 10 6 sperm/ml, using Tris egg-yolk medium (73% Tris base medium, 20% egg-yolk, and 7% glycerol; van Wagtendonk-de Leeuw, Haring, Kaal-Lansbergen, & den Daas, 2000). sperm was processed as described previously by Chaveiro, Machado, Frijters, Engel, & Woelders, (2006). Briefly, the diluted sperm was packed in 0.25 ml 'french' straws (IMV), at room temperature (24°C), and closed with a plug of polyvinylalcohol. The straws were placed horizontally in a water-filled (24°C) box, in a place at 4°C for 105 min (slow cooling and equilibration period). The cooled straws were exposed to liquid nitrogen (LN2) vapour for 8 min (4 cm above LN2 level, approximately −125°C to −130°C), plunged into LN2, and stored in LN2 for at least one week. The experiment was designed with 10 replicates and each replicate was performed on a separate sample. For each sample, the cauda epididymal sperm is cryopreserved in at least three straws (for sperm motility and gene expression patterns evaluation, and IVF).
Thawing was achieved by immersing the straws for 6 s in a water bath set at 65°C (Eriksson & Rodriguez-Martinez, 2000). Immediately after thawing, the sperm was processed for motility and molecular evaluations.

| RNA extraction and cDNA synthesis
The molecular assessment was performed on high and low motile sperm that were separated by centrifugation of fresh (control group) or frozen-thawed (treatment group) on a discontinuous Histoprep® (BAG Health Care) density gradient (0.5 ml 50% Histoprep over 0.5 ml 100% Histoprep) at 250 g for 7 min. After the centrifugation, the high motile sperm passed through the two gradients, while the low motile sperm passed only the 50% gradient. Total RNA was extracted from sperm using Iraizol reagent (RNA Biotechnology Company). Briefly, samples were lysed in 400 µl of this reagent and after 5 min mixed with 100 µl chloroform. The resulting mixture was centrifuged after 5 min (8000 g, 4°C, 5 min), yielding an upper aqueous phase containing total RNA. After addition of 500 µl absolute ethanol to the supernatant (20 min in −20°C), it was centrifuged (8000 g, 4°C, 5 min) and the RNA pellet was washed with 80% ethanol twice. The RNA samples were re-suspended in 20 ml DEPC-treated water and treated with RNase-free DNase (Sinaclon Bioscience) to avoid amplification of contaminating genomic DNA.
The amount and quality of RNA were determined by spectrophotometry (Amersham Pharmacia ultrospec 1100 Pro). Only RNA of sufficient purity, having an absorbance ratio (A260/280) greater than 1.9, was considered for the synthesis of cDNA.
Total RNA was reverse transcribed into cDNA in immediately after extraction (less than 2 hr) using M-MLV reverse transcriptase (Sinaclon Bioscience) as described by Nazari, Shirazi, Shams-Esfandabadi, Afzali & Ahmadi, (2016). The reverse transcription was done in a 20 µl volume containing 10 µl (14 µg) of extracted RNA and 1 µl random hexamer. This mixture was heated to 70°C for 5 min, and then 0.5 µl of RNase inhibitor, 2 µl RT buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2), 2 µl dNTP (10 mM) and 1 µl M-MLV reverse transcriptase were added. This mixture was incubated for 5 min at 25°C, followed by 60 min at 42°C. The mixture was heated to 70°C for 10 min to denature the RNA and then stored at −20°C.

| Quantitative real-time PCR
Real-time PCR was performed in two replicates for each sample (Rotor-Gene Q 6000). The GenBank accession numbers, sequences, the size of amplified products and annealing temperature of each primer are shown in Table 1. Half ml DNase I treated cDNA (containing 0.2 µg) was added to 6 µl of SYBR Premix Ex Taq II Mix and 0.75 µM of each specific primer in a total volume of 12 µl. The PCR programme was comprised of 45 cycles of 94°C for 40 s, 60°C-63°C for 30 s (annealing temperature; Table 1) and 72°C for 30 s.
Considering the selection of an appropriate housekeeping gene as a reference gene for normalization, several studies demonstrated that the Glut5 gene is highly reliable for analysis of relative gene expression in bovine fresh and frozen-thawed sperm samples (Ashish et al., 2017). Melt curve analysis was conducted to confirm the TA B L E 1 Sequence and annealing temperature of used primers for RT-PCR  (Ruijter et al., 2009). The following formula was applied to determine the relative gene expression in cloned embryos compared to the control group (IVF embryos; Dorak, 2007;Pfaffl, 2001).
The rate of blastocyst formation was recorded on days 7 and 8 after IVF. The cleavage and blastocyst rates are defined as the proportion of cleaved embryos and produced blastocysts per presumptive zygotes, respectively.

| Statistical analysis
The mean ± SEM differences in motility pattern and embryonic developmental rate between fresh and frozen-thawed sperm were analysed using independent samples t test. The relative abundance of gene expression between low and high motile sperm in fresh and frozen-thawed sperm groups was analysed using one-way analysis of variance (ANOVA) followed by Tukey post hoc test. All of the statistical analysis was performed by SPSS 20.0.0 software (IBM Corp.). Differences were considered significant at p < .05.

| RE SULTS
After cryopreservation and thawing of epididymal sperm, total motility of sperm and also slow progressive motility (class B; Figure 1), VCL, VAP, MAD, ALH and BCF (Table 2)  In assessment of abundance of transcripts encoding various motility-and fertility-related genes in epididymal sperm, it was shown that transcripts encoding TSSK6 were found to be affected by freezing especially in slow progressive motility status (p < .01) and their relative expressions were higher in fresh than frozen-thawed sperm ( Figure 2).
There was no difference between fertility related genes (PRM1 and PRM2) in high motile fresh and frozen-thawed sperm that were used in the IVF procedure. Freezing of sperm, however, decreased these fertility-related genes especially in low motile sperm (p < .01; Figure 2). Table 3, the cleavage rate did not present any significant differences between in vitro produced bovine embryos derived from fresh or frozen-thawed epididymal sperm. Furthermore, the type of sperm, fresh or frozen-thawed, had no effect on the overall blastocyst yield at days 7 (range 16.26%-18.05%) and 8 (range 21.77%-25.26%) among the two groups. No significant difference in each examined days (days 7 and 8) between the blastocysts rate of fresh and frozen-thawed sperm groups indicated that the developmental timing of bovine embryos to the blastocyst stage was not affected by the freezing of sperm.

| D ISCUSS I ON
In vitro production of embryo with cryopreserved recovery of the epididymal spermatozoa from dead animals is a useful tool to rescue genetic material that otherwise would be lost, either from highly productive livestock or endangered species. In this study, the Ratio = E GLUT5 (C t frozen thawed sperm)∕E (C t frozen thawed sperm) E GLUT5 (C t I fresh sperm)∕E (C t fresh sperm) F I G U R E 1 Percent motility (mean ± SEM) of prefreezed and post-thawed bovine epididymal sperm. a,b Refers to significant differences in the similar pattern columns(p < .05). Data represent n = 10 replicates spermatozoa from epididymis can be recovered with acceptable efficacy overnight after animal's death.
Cryopreservation processes are known as being damaging to the sperm cells due to changes during the biotechnological process, the toxic and osmotic stress depicted by exposure to cryoprotectants and the formation and dissolution of extracellular ice crystals (Januskauskas, Johannisson, & Rodriguez-Martinez, 2003;Watson, 2000). In addition to sperm motility, the sperm motility pattern, fertility potential and the pattern of gene expression can be considered as the sperm quality parameter. In this study, the effects of cryopreservation of epididymal bovine spermatozoa on velocity and the pattern of sperm motility, fertility potential and the expression panel of some motility (TSSK6) and fertility (PRM1 and PRM2)encoding genes viability pattern were also evaluated.
Reports in some ruminants such as bovine, goat and red deer in- epididymis stored at 4°C for a few hours before cryopreservation. In this study, testicles were refrigerated for a longer period than on the mentioned studies (Goovaerts et al., 2006;Nichi et al., 2017), without suffering more severe deleterious effects after freezing.
In cryopreservation, beside the moderate reduction of motility, the frozen-thawed epididymal sperm showed a more obvious decrease variables related to swimming velocity (VCL and VAP) with lower lateral displacements (MAD, ALH and BCF); whereas the linear trajectories of the sperm was not affected by cryopreservation (VSL, LIN, STR and WOB). In this study, the motility pattern of fresh and frozen-thawed sperm reflects the lack of any statistical differences in the developmental rate of bovine embryo-derived by fresh or frozen sperm, because the oocyte is probably fertilized with progressive sperm with lower lateral displacements (Jeulin et al., 1986).
Also, the significant difference in the velocity of the frozen-thawed sperm, which was seen only in slow progressive sperm, is justified by this reason, because those sperm with slow progressive motility have more lateral displacement that in the study was found in fresh sperm. Furthermore, cryopreservation negatively affected mitochondrial potential (Nichi et al., 2017). Since the mitochondria are TA B L E 2 Effect of freezing on the bovine epididymal sperm motility patterns using CASA in vitro  the main source of energy in the most mammal's sperm, the motion parameters changes related to lateral displacements of the sperm is because of impaired mitochondria following cryopreservation (Hung, Miller, Meyers, & VandeVoort, 2008). Therefore, in our study, The composition and abundance of spermatozoal transcripts vary between fresh and frozen-thawed bull sperm (Card et al., 2013;Selvaraju et al., 2018) probably due to degradation of mRNA during cryopreservation (Yeste, 2016) if appropriate measures are not implemented during vitrification/freezing or warming/thawing. For example, some studies showed that PRM1 (Ganguly et al., 2013) and DNMT3a and DNMT3B (Zeng et al., 2014) are affected during cryopreservation; exactly as we observed in our study about of given transcripts. In this study, abundance of TSSK6 transcripts was lower in post-frozen-thawed sperm especially in low motile sperm and their relative expressions were higher in fresh than frozen-thawed sperm. This is in accordance with a previous study showed that the transcripts encoding for a serine/threonine testis-specific protein kinase (TSSK6) were related to high-motility status in the bull and their relative expressions were higher in fresh than frozen-thawed sperm (Mondal, Baruah, Chatterjee, & Ghosh, 2013). But we did not show any difference between the transcription levels of this gene in both low and high motile sperm, either before or after freezing that its cause is unclear to us. However, the effect of cryopreservation on reducing the level of these transcripts was significant.
In accordance with the developmental rate, no differences were found in relative expressions of PRM1 and PRM2 genes between high motile fresh and frozen-thawed sperm that were used in IVF procedure. Freezing of sperm decreased these fertility-related genes in low motile sperm. In bovine, though PRM1 gene has high transcription and translation levels, the PRM2 has been reported to be transcribed and translated at low levels in spermatids (Maier, Nussbaum, Domenjoud, Klemm, & Engel, 1990) if at all, but absent in spermatozoa (Hecht et al., 2011). The levels of F I G U R E 2 Relative abundance of TSSK6 (A), PRM1 (B) and PRM2 (C) transcripts in bovine epididymal sperm derived overnight 4°C storage testes before and after freezing/thawing. mRNA was reverse transcribed, and subjected to real-time quantitative PCR. All reactions were normalized for glut5 mRNA expression. Values with superscripts ' a,b,c,d ' refers to significant (p < .05) differences in relative transcript abundance between groups PRM1 transcripts were significantly higher in good semen producers than poor counterparts, while the low transcription level of PRM2 did not differ between good and poor quality semen bulls (Ganguly et al., 2013). It should be emphasised that PRM1 and PRM2 genes are among the most strongly associated genes with different sperm quality parameters, such as morphology, motility and DNA integrity, as well as with fertility and finally embryo quality (Card et al., 2013(Card et al., , 2017Dadoune, 2009;Ganguly et al., 2013).
Evidence in this and other studies have been shown that the mRNA expression levels of PRM1 and PRM2 were significantly decreased in frozen-thawed bovine sperm (Ganguly et al., 2013), thus emphasising the results of different studies, and indicating that sperm DNA damage is induced by cryopreservation process (Castro et al., 2016;Gurler et al., 2016;Kopeika et al., 2015;Martin, Sabido, Durand, & Levy, 2004).
Beside the above-mentioned claims, the reduced levels of RNA in sperm cells during the cryopreservation may be due to changes in solution effect, ice crystal formation, free radicals, activation of the apoptotic and necrotic factors, pH and alteration in the membrane structure (Selvaraju et al., 2018). Although the mature spermatozoa are transcriptionally inactive (Grunewald, Paasch, Glander, & Anderegg, 2005), activation of the apoptotic factors during sperm cryopreservation (Martin et al., 2004) with the subsequent induction in expression of apoptosis-related genes such as caspases genes have been reported (Paasch, Grunewald, Agarwal, & Glandera, 2004).
However, the impact and mechanism of the cryo-induced spermatozoal transcript profiles are not yet understood and accordingly, their influence on fertility and embryo developmental competency remains to be elucidated. Additional studies would help to optimize the cryopreservation protocol for maintaining and stabilising the sperm functional capacity.
Although IVF techniques have been widely studied in cattle, few studies were found regarding the evaluation the potential of in vitro fertilizing of epididymal spermatozoa from dead bulls (Chaveiro et al., 2015;Martins et al., 2009;Martins, Rumpf, Pereira, & Dode, 2007;Nichi et al., 2017). Regarding IVF results, developmental rate of in vitro produced embryos using frozen/thawed epididymal sperm showed no significant differences in comparison with fresh counterpart. In terms of cleavage and blastocyst rate, the values are similar or more than those obtained by some studies (Chaveiro et al., 2015;Martins et al., 2007).
Despite the prominent effects of cryopreservation on bovine sperm cells, in vitro derived embryos with recovered spermatozoa from the cauda epididymal of recently slaughtered animals showed that spermatozoa can be obtained from the epididymis of either a highly productive farm animals or endangered species, which can be used for the production of viable embryos. It seems epididymides with providing the optimal environment for sperm storage in physiological conditions have adequate conditions to prolong sperm survival.
In this study, the blastocyst rate was recorded in two days (day 7 and 8) as a simple marker for developmental timing. Timing of embryo development has long been considered as an important marker to evaluate embryo quality. The speed of embryo development can be used a simple, rapid, precise and noninvasive method of embryo selection for transfer (Sallam, Sallam, & Sallam, 2016). Although the developmental timing of in vivo produced embryo usually proceeds according to a precise program in all species, in vitro produced embryos commonly show heterogeneity in their developmental timing and subsequently variability in developmental competence (Gutierrez-Adan, White, Van Soom, & Mann, 2015). The embryos with a timely manner development have the better developmental competence and viability after transfer, as shown in some studied species including human (Racowsky et al., 2000), rodents (McKiernan & Bavister, 1994) and cattle (Lonergan et al., 1999). Beyond the controversy surrounding the relation of developmental timing and embryo quality of bovine, no significant different blastocysts rate in each day (day 7 and 8) between the fresh and frozen-thawed sperm groups indicated that the developmental timing of bovine embryos to the blastocyst stage was not affected by the freezing of sperm.
In conclusion, our results indicate that (a) epididymal sperm had enough fertilizing potential and freezability even after overnight testes storage; (b) cryopreservation could affect the relative abundance of transcripts available in the sperm cell, but the percentage of in vitro produced embryo was not much affected; (c) cryopreservation changed the sperm motility pattern further in the direction of progressive movement with less lateral displacement.
The present study showed the potential for utilization of dead animal epididymal sperm of for preservation of valuable males.

ACK N OWLED G EM ENTS
This study was funded by a research grant of the corresponding author from Shahrekord University. The authors thank the Research Institute of Animal Embryo Technology for technical and financial supports, Shahrekord University.

CO N FLI C T O F I NTE R E S T
None of the authors have any conflict of interest to declare.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/vms3.355.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data are available on request from the authors.