Journal of the American Association for Laboratory Animal Science Copyright 2016 by the American Association for Laboratory Animal Science

Vol 55, No 5 September 2016 Pages 520–524

Cryotolerance of Sperm from Transgenic Rhesus Macaques (Macaca mulatta) Sean P Moran,1 Tim Chi,1 Melinda S Prucha,1,2 Yuksel Agca,3 and Anthony WS Chan1,2,* Cryopreservation is an important tool routinely used in preserving sperm for assisted reproductive technologies and for genetic preservation of unique animal models. Here we investigated the viability of fresh and frozen sperm from rhesus macaques on the basis of motility, membrane integrity, and acrosome integrity. Sperm motility was determined by visual evaluation; membrane and acrosome integrity were assessed simultaneously through triple staining with Hoechst 33342, propidium iodide, and fluorescein isothiocyanate–peanut agglutinin. We compared thawed semen that had been cryopreserved by using 2 different media with fresh semen from wildtype (WT) macaques; fresh semen from a model of Huntington disease (HD) with fresh WT semen; and fresh HD with cryopreserved-thawed HD semen. Our new freezing media (TEST EQ) preserved the acrosome better, with less net damage, than did traditional TEST (egg yolk extender containing TES and Tris) media. In addition, the percentage of membrane-damaged cells was similar in fresh HD semen (38.6% ± 2.9%) and WT semen (35.5% ±1.9%). Membrane and acrosomal damage were not different between HD and WT sperm after cryopreservation and subsequent thawing. Furthermore, cryopreservation had similar negative effects on the motility of HD and WT sperm. These data illustrate that semen from a rhesus macaque model of HD is similarly cryotoleratant to that from WT animals. Abbreviations: FM, freezing media; HD, Huntington disease; TALP–HEPES, Tyrode lactate–pyruvate–HEPES

Cryopreservation procedure is well known to cause numerous types of damage to the survival and integrity of the sperm.1 Damage to motility, mitochondria, tail structure and membrane and acrosome integrity can be found in frozen and subsequently thawed sperm.10 Cryoprotective agents are therefore added to the semen suspension to reduce some of this cellular damage caused during the freezing process.12 Increases in sperm cellular damage is directly correlated to a decrease in egg-fertilization ability in vivo.8 Although cryopreservation has been highly successful in agricultural animals, research in other mammals, especially exotic species, necessitates further work to enhance survival after thawing.13 The goals of the current study were to examine the effects of a new freezing media on survival after thawing of rhesus macaque semen and to explore the cryotolerance of a transgenic rhesus macaque model. Cryotolerance is defined as the ability of sperm to survive the rapid cooling and storage in liquid nitrogen and to maintain the ability to successfully fertilize an oocyte after thawing. Previous cryotolerance research has shown the TEST extender (an egg yolk extender containing TES and Tris buffers) to be the optimal freezing media for rhesus macaques.19 We have modified the TEST extender to contain iodixanol and the detergent Equex Paste (MiniTube, Tiefenbach, Germany), both of which have been shown to increase acrosome and membrane integrity after thawing in other mammalian species.2,9,17,22,24 With the TEST EQ extender confirmed as the better freezing extender, we then investigated differences in cryotolerance between our WT rhesus macaques and our transgenic Huntington disease (HD) animals.

Received: 16 Nov 2015. Revision requested: 11 Dec 2015. Accepted: 04 Mar 2016. 1Division of Neuropharmacology and Neurologic Diseases, Yerkes National Primate Research Center, Atlanta, Georgia; 2Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia; and 3Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri, Columbia, Missouri *Corresponding author. Email: [email protected]

The development of the rhesus macaque model of HD has been described previously.5,6,25 Only recently have the animals reached puberty and semen collection become possible, and freezing and subsequent banking of the semen from the 3 transgenic male macaques is of great importance. The ability to bank semen for later use is a critical aspect of any successful animal model, allowing for quick expansion of the cohort if needed. Previous research has shown that frozen rhesus sperm, used in conjunction with assisted reproductive technologies, results in live offspring.11 Before banking large quantities of semen, the quality of the HD semen needed to be assessed. While quantifying the effects of our new freezing media, we also were able to compare HD with WT rhesus sperm to evaluate the effects of disease status on sperm viability. In addition, we expect that the findings from our transgenic HD macaque model will provide insight into the fertility of postsymptomatic HD human patients.

Materials and Methods

Animal model. Transgenic HD macaques were generated as previously described.7 All HD and WT rhesus macaques (Macaca mulatta) were maintained at the Yerkes National Primate Research Center (Atlanta, GA). All animal procedures used in this study were approved by the IACUC of Emory University (Atlanta, GA). All documented research in this study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals.21 Chemicals and media. All chemicals were purchased from Sigma–Aldrich (St. Louis, MO) with the following exceptions: Tris (Fisher, Fair Lawn, NJ), propidium iodide (Invitrogen, Carlsbad, CA), and Equex Paste and 0.25-mL semen straws (MiniTube). Freezing media. TEST freezing extender media was prepared according to a published protocol19 and included 20% egg yolk, with pH of 7.4 and osmolality of 350 mOsm/kg. Briefly, this


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solution was centrifuged at 14,000 × g at 4 °C for 45 min and the pellet discarded. For our modified freezing media (TEST EQ), we added iodixanol (2%; OptiPrep) and Equex Paste (0.5%) to the supernatant before passing the solution through a 0.2-µm filter. TEST and TEST EQ extenders were aliquoted, flash-frozen, and stored at –80 °C. Animal handling and semen collection. A total of 5 ejaculates were collected by electroejaculation from 2 WT rhesus monkeys, to compare the freezing efficiency of the 2 freezing extenders TEST and TEST EQ. In the HD comparison study, the same 2 WT macaques and an additional WT male were compared with 3 HD animals. Each macaque was chair-trained by using a ‘pole-and-collar’ technique.4 The monkey was then lightly sedated with an intramuscular injection of ketamine (approximately 0.4 mg/kg body weight). One presized defibrillator gel electrode was wrapped around the base of the penis and connected to the negative electrode lead. The second gel electrode was positioned immediately behind the glans and connected to the positive lead. The device was slowly increased to 32 V or until ejaculation (whichever came first). When the sample was delivered, the date, time, peak output current, duration of electrical current, drug administered, behavioral observations, and technician initials were recorded. Ejaculate samples were kept at room temperature for 25 min to liquefy. The liquid sample was transferred into a fresh 15-mL conical tube and washed once with Tyrode lactate–pyruvate–HEPES (TALP-HEPES) medium supplemented with bovine serum albumin (4 mg/mL)19 and then centrifuged at 400 × g at room temperature for 5 min. The supernatant was discarded, and the sample was resuspended into 1mL total volume with TALP-HEPES containing bovine serum albumin. Evaluation of sperm motility. Motility was assessed visually by placing 10 µL diluted sperm suspension on a prewarmed microscope slide and covered with a 22-mm square cover glass. The slides were evaluated under a 10× positive phase objective on a light microscope. Motility was scored on a scale of 0 to 100 (minimum to maximum) by 3 independent observers blinded to freezing method and genotype. The individual scores were averaged together. Fluorescent staining. Sperm viability was quantified by using Hoechst 33342, propidium iodide, and fluorescein isothiocyanate–peanut agglutinin. Sperm samples were diluted to a concentration of 1.2 × 107/mL, aliquoted into a black microfuge tube containing 0.5 µM Hoechst 33342, and placed into a 37 °C water bath for 5 min. Subsequently, 2.0 µM propidium iodide was added and the sample incubated for another 5 min. Last, 4.8 µM fluorescein isothiocyanate–peanut agglutinin was added and the sample incubated for 20 min. Stained sperm were placed on a microscope slide coated with 0.1% poly-D-lysine (Sigma) and covered with a coated 22-mm square cover glass. A minimum of 2000 Hoechst-positive sperm were counted per sample. Cells stained with both propidium iodide (red) and Hoechst (blue) were considered to possess a damaged membrane and therefore deemed dead. Hoechst-positive cells with bright green fluorescence were considered to have a damaged acrosome (Figure 1 A). Viable sperm recovery (%) was calculated as:

Freezing protocol. The final concentration of the suspension containing freezing extender was 160 × 106 sperm/mL, so that each 0.25-mL straw contained approximately 40 × 106 sperm; the

Figure 1. Net acrosomal and membrane damage of WT rhesus macaque sperm in TEST extender as compared with TEST EQ extender. Whereas acrosomal damage after freezing was significantly (*, P < 0.05) less with TEST EQ compared with TEST, membrane damage did not differ between the 2 extenders.

freezing protocol used was described previously.19 The freezing extender was thawed and prewarmed to room temperature for 5 min before use. In brief, the total volume was divided equally between primary extender and secondary extender (6% glycerol added). First the primary extender was pipetted slowly into the resuspended sperm and mixed by gentle rolling to avoid damage. This sperm suspension was placed into a 1.0-L water bath at room temperature and then transferred into a 4 °C refrigerator. After 2 h, an equal volume of the secondary extender (4 °C) was gradually added to the sperm suspension over 10 min. The new suspension (3% total glycerol) was incubated at 4 °C for an additional 30 min. After the last incubation, the sperm suspension was loaded into 0.25-mL cryo-straws, which were sealed by using a commercially available straw sealer, placed on a custom holding rack, and transferred into a styrofoam box containing liquid nitrogen; the box contained enough LN2 so that the LN2 was 4 cm from the bottom of the straw-holding rack. The box was covered and incubated for 8 min, during which enough LN2 was added at 2-min intervals to raise the LN2 level in 1-cm increments, until the bottom of the rack of straws was 1 cm above the LN2. The straws were then immersed in LN2 and transferred into LN2 tanks for long-term storage. Thawing of cryopreserved sperm. Sperm straws frozen in LN2 were allowed to thaw in a 37 °C water bath for approximately 30 s, washed with 2 mL of 37 °C TALP-HEPES medium supplemented with bovine serum albumin, centrifuged at 400 × g for 5 min at room temperature, and the supernatant discarded. The pellet was resuspended by gentle pipetting to a concentration of 1.2 × 107/mL with TALP-HEPES containing bovine serum albumin. Motility and fluorescence were scored and recorded as described earlier. Comparison of freezing media. Each fresh semen ejaculate was divided in half by volume, with one half frozen by using TEST media as the primary extender and the other half frozen by using TEST EQ according to the described protocol. Sperm samples were evaluated after a minimum of 1 week of storage in LN2. The extender that provided the better sperm viability and motility after thawing was used in the HD comparison study. In comparing damage between WT and HD sperm, a total of 14 ejaculates were collected from 3 WT macaques and 5 ejaculates from HD macaques. To assess percentage net damage, 9 ejaculates from 3 WT and 5 ejaculates from 3 HD macaques were used. Oocyte retrieval and intracytoplasmic sperm injection. Oocytes were collected according to a published procedure.7 In brief, ovulation was induced in a female rhesus monkey exhibiting regular menstrual cycles by using exogenous gonadotropins. Follicular aspiration was performed by using a suction device attached to a 20-gauge stainless steel hypodermic needle; oocytes were collected into a sterile 15-mL conical tube containing TALP–HEPES supplemented with 5 IU/mL heparin (Sigma) 521

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to prevent blood clots during aspiration. After aspiration, the oocytes were prepared for in vitro fertilization by intracytoplasmic sperm injection of thawed cryopreserved sperm.7 Fertilized embryos were cultured until they reached blastocyst stage. Data analysis. Sperm viability and motility were analyzed by one-way ANOVA. Freezing extender comparison and semen comparison between WT and HD macaques were performed independently by a statistician who was blinded to experimental conditions and using the statistical software program JMP (SAS Institute, Cary, NC). Results were considered significantly different when the P value was less than 0.05.


Effect of freezing extender on cryopreservation of sperm from WT rhesus macaques. Average net damage in the membrane and acrosomal integrity assays was determined for a total of 5 ejaculates from 2 WT male macaques. Net acrosomal damage (mean ± 1 SD) was significantly (P < 0.006) greater with the TEST extender (40.8% ± 3.1%) compared with the TEST EQ group (28.6% ± 3.0%; Figure 1). Membrane damage did not differ between the 2 groups, according to one-way ANOVA. Cryopreservation of sperm from HD rhesus macaques. Because TEST EQ was the more efficient extender with regard to acrosome integrity, we chose it for the HD study. Each ejaculate from HD macaques was split into fresh and frozen samples and evaluated individually for membrane and acrosomal damage. In addition, the cryopreservation data from 3 transgenic HD monkeys were combined and compared with results pooled from 3 age-matched WT controls. Both WT and HD groups showed increased damage after as compared with before freezing (Figure 2). However, acrosomal and membrane damage after cryopreservation did not differ between WT and HD sperm. Motility of WT sperm compared with HD sperm. Sperm motility was assessed through blinded scoring by 3 independent expert observers. Scores were averaged and pooled into HD and WT groups. Motility declined after cryopreservation in both HD semen (before freezing, 89.3% ± 3.5%; after freezing, 63.5% ± 11.8%, P < 0.001, n = 7) and WT samples (before freezing, 83.96% ± 4.37%; after freezing, 51.3% ± 7.0%, P < 0.001, n = 10). However, cryotolerance in terms of motility was not significantly different in HD semen compared with the WT group (Figure 3). Fertilization using cryopreserved sperm. Sperm from a transgenic HD male rhesus macaque was cryopreserved for at least 1 wk and then thawed according to the described protocol. Thawed sperm successfully fertilized rhesus macaque oocytes from a WT oocyte donor, as shown by the formation of 2 pronuclei (Figure 4). A total of 7 oocytes were fertilized by using the thawed sperm sample, and 2 (28.6%) reached blastocyst stage at day 8 (Figure 4). Although few oocytes were fertilized, the overall embryo development rate is comparable to our prior reports.16,20 These data suggest that cryopreserved HD macaque sperm retain fertilization capacity.


Data comparing sperm viability between healthy men and that of men with HD are unavailable currently. This study presents new data in which 2 freezing extenders were compared directly and in which the sperm from 3 transgenic HD rhesus macaques was compared with samples from 3 WT rhesus sperm donors. TEST EQ was the better freezing extender for cryopreserving sperm and led to less acrosomal damage than did the TEST extender. However, cryopreservation method is not free of deleterious consequences.23 Although the cryotolerance of the sperm in terms of acrosomal damage was increased

Figure 2. Membrane and acrosome integrity of frozen–thawed sperm. Representative images of rhesus macaque sperm stained with (A) fluorescein isothiocyanate–peanut agglutinin-positive sperm (green), (B) propidium iodide (red), and (C) Hoechst 33342 (blue), and (D) an overlay image of panels A through C. (E) WT (15 total samples from 3 macaques) and HD (7 total samples from 3 macaques) semen did not differ with regard to the membrane or acrosomal integrity of individual samples. (F) Net percentage damage did not differ between WT and HD groups with regard to membrane or acrosomal integrity.

Figure 3. Motility of fresh sperm did not differ between WT (10 total samples from 3 macaques) and HD (7 total samples from 3 amimals). In addition, although the motility of cryopreserved sperm was significantly (*, P < 0.05) decreased in both WT and HD samples, motility did not differ between cryopreserved HD and WT sperm.

by using TEST EQ, the overall viability of cryopreserved sperm (on the basis of motility and membrane and acrosomal integrity) is still less than that of fresh sperm. That being said, the decreased viability after cryopreservation did not completely prevent fertilization (Figure 4). Although motility was decreased nearly 50%, which was suboptimal, assisted reproductive technologies are available to overcome the lowered sperm motility. The sperm’s ability to successfully fertilize an oocyte that subsequently develops into a blastocyst in vitro confirms that the sperm’s fertilization capacity intact. However, whether frozen macaque semen can be used for artificial insemination has yet to be determined. Improvements in storage and preservation techniques for rhesus macaque sperm can benefit endangered species of NHP as well as animals, such as our HD macaques, with unique genetic makeups or desired traits. Research into sperm


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Figure 4. In vitro development of embyos fertilized by using frozen–thawed HD monkey sperm. Cryopreserved sperm from an HD rhesus macaque was thawed and used in intracytoplasmic sperm injection to fertilize WT rhesus maqes oocytes. Images were captured during in vitro culture of live embryos after fertilization using Olympus inverted microscope. Overall embryo development rate is comparable to prior reports. (A) Zygote with 2 pronuclei (day 1). (B and C) Morula stage (day 4). (D) Blastocyst (day 8). Scale bar, 20 μm.

cryotolerance is important for the establishment of a sperm bank and future derivation of the rhesus macaque HD model. This study is the first to illustrate the similar cryotolerance of HD and WT rhesus macaque sperm. The abilities to expand the model cohort as needed for preclinical research use14 and to distribute the cryopreserved sperm to other research facilities are critical for future preclinical and biomedical applications of the HD macaque model. The HD macaque model is one of many HD animal models, including those involving miniature pigs, sheep, and rodents.3,5,15,18,25 Each model has its strengths, but none provides a perfect recapitulation of the human disease. For example, the minipig model shows a 75% decline in sperm motility in transgenic HD group compared with WT animals.3 In contrast, our HD macaque model does not show any significant difference in sperm viability compared with WT controls. HD macaques develop progressive declines in cognitive behavioral functions and neuroanatomical changes similar to those observed in humans, without a loss in fertility, suggesting the potential use of HD macaques as a preclinical large-animal model.5,6 Further research in sperm viability, as the disease progresses in our animals, may lead to new insights regarding fertility in human patients with HD. Interestingly, no problems regarding sperm quality or fertility in human HD patients have been reported, but no patients with late-onset or late-stage disease have been evaluated. Continued research is needed to determine whether a decline in motor function correlates with a decline in fertility in HD rhesus macaques. Our HD model, in which its fertility during the early onset of HD symptoms has been evaluated, can be used for testing the efficacy of potential HD treatment strategies and their effects on fertility in humans. Our findings improve current cryopreservation methods, but further optimization of HD sperm cryopreservation is necessary to improve survival after thawing, which is essential for the establishment of a sperm bank. In addition, our study suggests that HD macaques might be a useful model for optimizing sperm cryopreservation for human HD patients.


We thank the veterinarian and animal care staff of Yerkes National Primate Research Center for providing outstanding services. YNPRC is supported by the Office of Research and Infrastructure Program (ORIP)/OD P51OD11132. This study is supported by grant awarded by the ORIP/NIH (RR018827) to AWSC.


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Cryotolerance of Sperm from Transgenic Rhesus Macaques (Macaca mulatta).

Cryopreservation is an important tool routinely used in preserving sperm for assisted reproductive technologies and for genetic preservation of unique...
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