Cryopreservation of Mauritian Cynomolgus Macaque (Macaca fascicularis) Sperm in Chemically Defined Medium

Abstract

The objective of this study was to optimize cryopreservation of sperm from Mauritian cynomolgus macaques (MCM) in defined conditions. Sperm viability and motility were compared between sperm cryopreserved in chemically-defined freezing media with variable osmolarity and the presence of either ethylene glycol or glycerol. The highest percentage viability (after freeze-thaw) was seen in sperm samples that were cryopreserved in medium with an osmolarity of 310 mOsm, while higher osmolarities markedly decreased sperm viability. Ethylene glycol and glycerol at concentrations of 4.6% and 5%, respectively, preserved sperm viability to an equivalent degree. Although higher motility rates and higher straight-line velocities were observed in sperm samples frozen in glycerol compared with ethylene glycol, these differences were not statistically significant. Thawed sperm frozen in defined conditions with glycerol were capable of fertilizing MCM oocytes in vitro, with development to the blastocyst stage. The protocol described here provides an effective method for cryopreservation of sperm to facilitate subsequent in vitro fertilization and genome editing of embryos in MCM species.

Mauritian cynomolgus macaques (MCMs) are a subspecies of Macaca fascicularis that are descended from a small founder population and as such, have a very limited MHC diversity consisting of only 7 common haplotypes (M1-M7).³ This unique feature makes them an exceptional source of research animals for the investigation of AIDS pathogenesis, novel vaccine development, and stem cell therapies.²² Recent advances in genetic engineering of nonhuman primates (NHP) has opened opportunities to use a macaque model for studies of AIDS, by engineering animals with genetic modifications which affect molecular pathways involved in retroviral infection.¹³ Cryobanking of MCM sperm could provide an effective way to preserve these valuable genetic resources, facilitate in vitro fertilization (IVF) experiments, and allow the application of genome editing strategies in these MCM embryos.

Mammalian sperm cryopreservation is often accompanied by a decrease in sperm quality, motility, survival, and the sperm's ability to fertilize. Osmotic and cold shock, and the formation of intracellular ice crystals are the leading damaging factors. Collectively, the associated damage initiates oxidative stress, leading to a decrease in the fertilizing ability of sperm, as reviewed elsewhere.¹¹ The formulation of the extender, cryopreservation agents and the cooling and thawing rates significantly impact the outcomes for successful sperm cryopreservation.

Sperm preservation protocols vary highly between species due to inherent characteristics such as sperm size, morphology, phospholipid membrane composition, and metabolism. Although numerous approaches to freezing sperm from cynomolgus macaques have been described,^{14-16,23,24,26,28} the conditions for freezing sperm from MCMs specifically has not been evaluated. In addition, the currently available freezing media formulations for macaque sperm commonly contain egg yolk in the extender as a source of lipoprotein to protect the sperm membrane against cold shock.^{8,15,18,21,24-26} However, the use of egg yolk often causes sperm agglutination, increases the risk of contaminating sperm with viruses or other pathogens from the egg yolk, complicates sperm manipulation during intracytoplasmic sperm injection (ICSI), and makes it difficult to maintain consistency in the freezing procedure.^1,2,16

The goal of the present study was to develop a protocol for freezing MCM sperm using a chemically defined extender. The functionality of sperm recovered after freeze-thaw was assessed by analyzing sperm survival via flow cytometry, sperm kinematics via computer-assisted sperm analysis (CASA), and in vitro fertilization potential via ICSI of MCM oocytes.

Materials and Methods

Animals.

Cynomolgus macaques (Macaca fascicularis) of Mauritian origin were acquired from Bioculture Mauritius, or Covance Research Products. Four sexually mature males, ages 5 to 11 y, weighing 7 to 9 kg, and 7 regularly-cycling females, ages 5 to 12 y, weighing 3.5 to 7 kg were used in this study. All procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and under the approval of the University of Wisconsin College of Letters and Sciences and Vice Chancellor Office for Research and Graduate Education Institutional Animal Care and Use Committee.

Semen collection.

Sperm was collected from male macaques by penile electrical stimulation while under ketamine sedation. Ejaculates were incubated at 37 °C for 20 min to allow for liquifaction. Typically, the liquified ejaculate volume ranged from 0.1 to 0.3 mL. A 10 mL volume of warm (37 °C) semen extender (see Table 1) without cryoprotectant was added to the ejaculate. After gentle mixing, diluted samples were passed through a 70 μm strainer. For evaluation of fresh sperm samples, 1 mL of the sperm suspension was removed and diluted with 3 to 5 mL of human sperm washing medium (Irvine Scientific, cat no. 9983). The remaining 9 mL of the sperm suspension was centrifuged at 350 × g for 8 min, and 8 mL of the clear supernatant was removed. The sperm pellet within the remaining 1ml then used for freezing.

Table 1.

Composition of extender solution

Ingredients	Final Concentration	Amount
Trehalose	58 mM	2.63 g
PIPES	16 mM	598 mg
TES	52 mM	1,429 mg
Raffinose	4 mM	3.7 g
Glucose	111 mM	1.123 g
Proline	5 mM	72 mg
Glycine	10 mM	90 mg
Glutamine	10 mM	72 mg
30% human albumin	9.1%	36 mL
Total Volume		120 mL
Osmolarity	310mOsm	N/A
pH	7.2-7.4	N/A

Sperm freezing and thawing.

One mL of precooled extender (4 °C, Table 1) with 2× concentration of cryoprotectant (Table 2) was added dropwise to the 1 mL of the sperm suspension every 3 to 4 min within 30 min, while the tube was kept on wet ice.²⁹ Sperm samples were then aliquoted into PCR tubes (VWR, cat no. 93001 to 118) (approximately 90 μL/each) and incubated at 4 °C for 45 to 60 min. These tubes were then placed approximately 3 cm above the surface of liquid nitrogen (LN) for 1 to 2 h and subsequently transferred into vapor phase LN for permanent storage at -160 °C. Sperm samples were thawed by actively warming tubes in a 37 °C water bath for 20 s, then transferred into a 4 mL tube (Falcon, cat no. 352054) followed by dilution with 3 mL human sperm washing medium and centrifuged at 350 × g for 8 min.

Table 2.

Cryoprotectants added to extender solution for freezing MCM spermatozoa

Cryoprotectants	Percent	Molarity	Volume added to 10 mL of extender
Glycerol 2× v/v	10%	1.4 M	220 µL
Ethylene Glycol 2× v/v	9.2%	1.5 M	180 µL

Freezing Extender.

The freezing extender was formulated with piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES) and 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES) buffering agents, trehalose, raffinose, glucose, and 3 amino acids (proline, glycine, glutamine) beneficial in cryopreservation of sperm in cynomolgus macaques,¹⁴ and human albumin (Table 1). Two cryoprotective agents, glycerol or ethylene glycol (Sigma–Aldrich), were evaluated at concentrations of 2% to 7% and 1.5% to 6.5%, respectively (Table 2). The osmolarity of the extender was modified by changing the concentration of carbohydrates and assessed using a 3300 Micro–Osmometer (Advanced Instruments), based on freezing point. Osmolarities ranging from 310 to 455 mOSM were assessed. Extender, containing either glycerol or ethylene glycol, was initially added at twice the concentration to achieve the desired concentration of cryoprotective agents after 2-fold dilution with sperm suspension.

Evaluation of sperm viability and motility.

Viability and motility of fresh and frozen-thawed sperm samples were analyzed by flow cytometry and CASA, respectively. For evaluating sperm viability by flow cytometry,¹⁰ sperm samples were washed with human sperm washing medium (Irvine Scientific) and diluted to a concentration of 5 × 10⁶ - 10 × 10⁶ cells/mL. A volume of 0.5 μl SYBR-14 and 5 μl propidium iodine (PI) (Thermofisher LIVE/DEAD Sperm Viability Kit, cat no. L-7011) was added to 1 mL of diluted sperm and incubated for 10 to 15 min at 37 °C. The samples were then run on a MACSQuant flow cytometer (Miltenyi Biotech) and data were analyzed using FlowJo software Version 7.0 (BD; www.flowjo.com). Three ejaculates, collected at 1 to 2 wk intervals from a single animal, were used for this assay. Approximately 100,000 to 150,000 sperm were analyzed per sample.

Sperm motility was evaluated by CASA, using an IVOS II system (Hamilton-Thorne Biosciences). Semen samples were diluted with prewarmed (37 °C) human sperm washing medium, to achieve a concentration not exceeding the operational range of the CASA system (<60 × 10⁶ sperm cells/mL). Counter chambers (Leja) were filled with 3 μl diluted sperm and 3 fields, containing 10 to 40 sperm per field, were captured. Sperm kinematic parameters assessed by CASA included motility, progressive sperm motility, average path velocity (VAP), curvilinear velocity (VCL) and straight-line velocity (VSL). Sperm samples from 4 individual males were analyzed before and after freezing in either glycerol or ethylene glycol. Four sperm samples per animal were collected at a 1 to 2 wk interval.

Mauritian cynomolgus macaque embryo culture.

Oocytes were obtained using methods previously described for rhesus macaques at the WNPRC,^5,27 with some modifications. Ovarian hyperstimulation was initiated on days 1 to 4 of menses, and achieved by administering 30 IU of recombinant human follicle stimulating hormone (FSH; follitropin β; IVFPrescriptions, Puregon) twice-daily, by intramuscular injection, for 11 to 12 d. A single injection of 1,000 IU of human chorionic gonadotropin (hCG) (IVF Prescriptions, Ovidrel) was administered at 1900 h on day 11 or 12. Oocytes were retrieved by laparoscopic aspiration between 38 to 40 h after the hCG injection. After transition from germinal vesicle (GV) and metaphase I (MI) to metaphase II (MII) stage (as morphologically confirmed by visualization of an extruded polar body), mature oocytes were fertilized by ICSI and cultured in global total medium (CooperSurgical Fertility and Genomic Solutions, cat no. LGGT-060).

Statistical analysis.

All data are expressed as mean ± SD. Cell viability was assessed using a one-way ANOVA test followed by a Bonferroni-Holm correction for multiple comparisons between groups using the Online Web Statistical Calculators at astatsa.com. Sperm motility, progressive motility, and sperm kinematic parameters were analyzed by one-way ANOVA and Fisher least significant difference (LSD) test using Microsoft Excel software.

Results

The major purpose of extenders is to protect sperm from their own toxic byproducts and to remove inorganic ingredients that negatively affect cryopreservation (for example, sodium chloride).²⁶ Previously described extenders for freezing macaque sperm^{8,15,18,21,23,25,29} contain egg yolk and Tris buffers, which change pH at low temperature and are thought to contribute to reduced sperm survival rates. To overcome these issues, we designed an extender composed of PIPES and TES buffering agents, trehalose, raffinose, glucose, amino acids and human albumin (Table 1).

Given that osmolarity is also major factor affecting cryopreservation,²⁴ we tested how modulating extender osmolarity affected sperm freezing in the presence of 5% glycerol. Sperm viability was measured by flow cytometric analysis with PI and SYBR-14 (Figure 1 A). We found that an osmolarity of 310 mOsm provided the best sperm viability after thawing, while a higher osmolarity markedly decreased sperm viability (Figure 1 B).

Figure 1.

Evaluation of sperm viability and motility after cryopreservation. (A) representative flow cytometry plot shows staining of MCM sperm with SYBR-14 and Propidium iodine (PI). Left dot plot shows forward compared with side scatters; right dot plot shows PI compared with SYBR-14 staining. Live sperm population negative for PI and positive for SYBR-14, moribund sperm population positive for SYBR14 and PI; and dead sperm population positive for PI and negative for SYBR14. The FSC/SSC gating we used excludes debris. Plotting PI vs SYBR14 without gating increases proportion of SYBR14negPIneg cells, but has a minimal effect on ratio of viable/dead cells. (B) Effect of osmotic pressure cryoprotectant solution on thawed sperm viability rate. Isotonic solution was used as control for fresh sperm. (C) Effect of ethylene glycol concentration in cryoprotectant solution on thawed sperm viability rate. (D) Effect of glycerol concentration in cryoprotectant solution on thawed sperm viability rate. Values in (B)-(D) are expressed as the mean ± SD for 3 ejaculates from one animal. No significant differences are present between bars marked with the same letter. A significant difference exists between bars marked with different letters (P < 0.05).

Next, we evaluated the effect of cryoprotectants on sperm viability using either ethylene glycol (1.5% to 6.5%) or glycerol (2% to 7%), added to a sperm extender with an osmolarity of 310 mOsm. We chose these concentrations based on previous reports determining that 3% to 5% glycerol and ethylene glycol were the optimal concentrations for these cryoprotectants in sperm freezing media used for rhesus and cynomolgus macaques.^{6,7,15,16,21,24-26,29} We found that concentrations of 4.6% ethylene glycol and 5% glycerol resulted in a higher degree of MCM sperm viability of 60.3% and 60.8% (Figure 1 B and 1 C), respectively. These data suggest both cryoprotectants are suitable for freezing MCM sperm.

The importance of sperm kinetics as a factor in fertilization success has been demonstrated in early studies,^17,30 but poor sperm recognition in early computerized systems made it unreliable.¹⁹ However, the effectiveness of automated determination of sperm motility parameters in predicting pregnancy outcomes after intrauterine insemination in humans has been confirmed using contemporary sperm motility analyzers.^4,9 We used a Hamilton-Thorne IVOSII CASA system to characterize sperm kinetics of 4 individual semen samples collected from 4 animals (Figure 2). Variations in sperm motility and progressive motility of sperm frozen with ethylene glycol compared with glycerol were not statistically different (Table 3). Assessing sperm velocity parameters revealed that both cryoprotectants increased VCL (Table 4), which is a negatively affected by exposure to cold stress.²⁰ The effect of freezing on other sperm kinetic characteristics (VAP and VSL) was minimal and insignificant. Comparison of velocity parameters of sperm frozen with glycerol or ethylene glycol did not reveal statistically significant differences in VAP and VCL parameters between the 2 cryoprotectants. However, sperm frozen in glycerol had slightly higher VSL values, which has been considered a most significant parameter in evaluating the effects of freezing on sperm function.²⁰

Figure 2.

Representative image of the CASA IVOS II capture field with individual sperm movement tracking. Sperm tracks are denoted as follows: red squares show static/nonmotile sperm, green tracks denote motile sperm, yellow denotes late tracking of the sperm, and turquoise denotes progressive motile sperm.

Table 3.

Comparison of motility of sperm frozen with different cryoprotectants.

Cryoprotectant	Motility (%)	Progressive Motility (%)
Control Fresh Sperm	59.9 ± 12.8	48.8 ± 12.3
Glycerol 5%	51.6 ± 9.9	43.0 ± 9.4
Ethylene Glycol 4.6%	42.0 ± 11.9	36.4 ± 8.4

Values are expressed as mean± SD of 16 ejaculate samples from 4 animals.

Table 4.

Sperm kinetic values prior to freezing and following freeze-thaw with different cryoprotectants.

	VAP (μm/s)	VCL (μm/s)	VSL (μm/s)
Control Fresh Sperm	133.3 ± 43.9	127.55 ± 17.3	123.7 ± 40.2
Glycerol 5%	129.3 ± 33.2	201.0 ± 19.7*	116.7 ± 21.2
Ethylene Glycol 4.6%	128.3 ± 51.9	197.1 ± 34.6*	97.7 ± 49.8

Values are expressed as mean ± SD 16 ejaculate samples from 4 animals were examined.

^* P < 0.05 as compared with control fresh sperm.

To confirm that frozen-thawed sperm retain their fertilization potential, frozen-thawed sperm samples (stored frozen for 20 to 56 wk) were used to fertilize MCM oocytes by ICSI. An ICSI procedure was implemented because we found that MCMs had a low fertilization rate as compared with domestic origin cynomolgus macaques after conventional gamete coculture (unpublished data), even with fresh semen. Similar to prior investigations that used sperm from Chinese cynomolgus macaque frozen in medium containing egg yolk,⁶ we did not observe significant differences in sperm viability in MCM sperm samples frozen in our chemically defined extender with glycerol or ethylene glycol. However, we decided to use sperm cryopreserved with glycerol for ICSI studies due to the improved sperm motility observed in samples frozen in glycerol compared with ethylene glycol. Moreover, glycerol was selected as the cryoprotectant for evaluation of fertilization potential because ethylene glycol can potentially cause embryotoxicity.³¹ Successful fertilization was achieved in 12 out of 15 oocytes (80%), as shown by the formation of 2 pronuclei. Six of them underwent cellular cleavage, and one oocyte reached the blastocyst stage by day 7 (Figure 3). This data confirms that frozen MCM sperm retain fertilization capacity, even after freezing with glycerol as a cryoprotectant.

Figure 3.

MCM embryo development following fertilization by ICSI. (A) Image displays an MCM oocyte undergoing fertilization by ICSI. (B) A representative image of an MCM blastocysts derived from an oocyte fertilized by a cryopreserved sperm.

Discussion

Successful freezing of cynomolgus monkey sperm has previously been achieved using a home-made egg yolk- and TEST-based medium containing glycerol.^24,26 However, the use of this media increases the risk of contaminating sperm with viruses or other pathogens from the egg yolk. Although commercial freezing media containing egg yolk reduces the possibility of such contamination, we found that these media still complicate the ICSI procedure. Components of egg yolk are retained in the sperm pellet even after several cycles of washing; these components can cause sperm agglutination, may clog the ICSI micropipette, and obscure sperm visibility, making the ICSI procedure more difficult. Moreover, multiple centrifugations during washing can severely damage sperm, causing tail banding and self-twisting.

Compared with the commercial defined extender used for freezing Chinese cynomolgus monkey sperm,¹⁶ our defined extender is based on PIPES and TES, giving it a stronger buffering capacity. This defined extender also includes 3 amino acids (proline, glycine, glutamine) known to be beneficial in cryopreservation of sperm¹⁴ and a protein-stabilizing osmolyte, trehalose.¹² These modifications improved motility recovery rates.

Here, we have demonstrated for the first time the successful cryopreservation of, and in vitro fertilization with, MCM sperm frozen in defined conditions using ICSI. Although sperm motility is less of a concern when employing ICSI, evaluation of motility parameters is still critical to achieving a high fertilization rate with frozen sperm, since any reduction in the number of motile sperm decreases the rate of fertilization and the recovery of good quality embryos after ICSI.³²

Overall, our studies revealed that viability and motility parameters of MCM sperm frozen in defined conditions were similar to those of sperm from rhesus and cynomolgus macaques frozen with egg yolk-containing extenders^{8,15,18,21,24-26} and much better than those of cynomolgus sperm frozen in commercial egg yolk-free medium.²⁸ MCM sperm that was cryopreserved in chemically defined extender retained fertilization capacity in vitro. The described freezing protocol can therefore be used to create a MCM sperm cryobank and as a model for genetic engineering.