Trehalose improves rabbit sperm quality during cryopreservation

Accepted Manuscript Trehalose improves rabbit sperm quality during cryopreservation Zhendong Zhu, Xiaoteng Fan, Yang Pan, Yinghua Lu, Wenxian Zeng PII:

S0011-2240(16)30472-2

DOI:

10.1016/j.cryobiol.2017.02.006

Reference:

YCRYO 3818

To appear in:

Cryobiology

Received Date: 13 December 2016 Accepted Date: 17 February 2017

Please cite this article as: Z. Zhu, X. Fan, Y. Pan, Y. Lu, W. Zeng, Trehalose improves rabbit sperm quality during cryopreservation, Cryobiology (2017), doi: 10.1016/j.cryobiol.2017.02.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Trehalose

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improves rabbit sperm quality during cryopreservation

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Zhendong Zhu1; Xiaoteng Fan1; Yang Pan1; Yinghua Lu1; Wenxian Zeng1

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712100, China

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Northwest A&F University, No.22 Xinong Road, Yangling, Shaanxi 712100, China.

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Tel: +86 (029) -87091932;

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Fax: +86 (029) - 87091932;

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E-mail: [email protected]

College of Animal Science and Technology, Northwest A&F University, Shaanxi,

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Corresponding author: W Zeng, College of Animal Science and Technology,

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Abstract High levels of reactive oxygen species are associated with spermatozoa

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cryopreservation, which bring damage to functional spermatozoa. The aim of the

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present study was to investigate whether and how the freezing extenders

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supplemented with trehalose was beneficial for the survival of rabbit spermatozoa.

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semen was diluted with Tris-citrate-glucose extender addition of different

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concentrations of trehalose. Addition of 100 mM trehaose significantly improved

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post-thaw rabbit sperm parameters, such as motility, intact acrosome, membrane

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integrity and mitochondrial membrane potential. Moreover, when freezing extenders

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supplemented with trehalose, activities of catalase (CAT), superoxide dismutase (SOD)

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and total antioxidant capacity (T-AOC) of post-thaw spermatozoa were enhanced,

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meanwhile, reactive oxygen species (ROS) level and Malondialdehyde (MDA)

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content were decreased. The results suggest that freezing extenders supplemented

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with 100 mM trehalose resulted in less ROS level and MDA content, higher motility

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and mitochondrial membrane potential as well as the integrity of acrosome and

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plasma membrane. Supplementation of trehalose with freezing extenders is beneficial

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to the rabbit breeding industry.

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Key words: antioxidants; cryopreservation; rabbit; semen; trehalose

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Introduction Cryopreservation of spermatozoa is an important technique for preservation of male fertility. However, the cryopreserved sperm has not been applied in rabbit

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breeding industry owing to the lower survival and fertility, smaller litter sizes and

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higher production cost [23]. High levels of reactive oxygen species is produced during

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cryopreservation, which is detrimental to spermatozoa, resulted in loss of motility and

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damages of structural membrane [8,33]. Thus, supplementation of antioxidants with

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freezing extender may protect spermatozoa against oxidative stress to improve the

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quality of post-thaw sperm.

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Trehalose, known as α-D-glucopyranosyl α-D-glucopyranoside, is a non-reducing disaccharide of glucose. Trehalose could be utilized to stabilize simple systems, such

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as lipids and proteins, as well as more complex biologicals [25]. As trehalose could

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reduce intracellular ice crystal formation and maintain the protein structural during

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sperm cryopreservation [20], it has been used for improving the quality of post-thaw

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spermatozoa in mammals animals including rams [4], boars [13], goats [1], bulls [30].

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Dalimata and Graham (1997) [11] reported a significant improvement in rabbit sperm

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quality when sperm were frozen in an egg yolk-acetamide extender supplemented

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with trehalose. However, Kozdrowski et al.(2009) [18] stated that addition of

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trehalose to the extender did not have a favourable effect on viability and fertility of

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post-thaw European brown hare spermatozoa. Thus, it is unclear whether the freezing

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extenders supplemented with trehalose is beneficial to post-thaw rabbit sperm or not.

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The aim of the study was to assess whether and how the trehalose protect rabbit sperm

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during cryopreservation. In our previous study [33], ROS was accumulated during cryopreservation and post-thaw incubation in rabbits. Unfortunately, spermatozoa are rich in

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polyunsaturated fatty acids which is susceptible to lipid peroxidation, and Tuncer et al.

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(2013) [31] reported that trehalose protected spermatozoa by reducing lipid

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peroxidation. Therefore, we hypothesized that exposed of rabbit spermatozoa to

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trehalose before freezing could improve the post-thaw spermatozoa quality by its

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antioxidative capacity. The present study was conducted to examine (i) activities of

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catalase, superoxide dismutase and T-AOC of post-thaw spermatozoa; (ii) intracellular

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ROS, MDA content of post-thaw spermatozoa; (iii) motility, membrane integrity,

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acrosome intactness, mitochondrial membrane potential of post-thaw spermatozoa.

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Materials and methods

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Animals Selection

All experimental procedures involving animals were approved by the Northwest

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A&F University’s Institutional Animal Care and Use Committee. seventeen mature

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rabbits (fifteen males, two females) were selected for the present study. Rabbits were

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individually housed and maintained under uniform feeding and managemental

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conditions, and fed in a commercial standard diet and water ad libitum.

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Semen collection and evaluation

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An artificial vagina was used to collect semen twice for a week. Each ejaculate was shifted to water bath maintained at 37oC and sent back to the Lab less than 30

ACCEPTED MANUSCRIPT min for evaluation. Sperm concentration of each sample was evaluated by

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hemocytometer. Motility was assessed with a phase-contrast microscope (Olympus,

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Japan) at 400× magnification. Ejaculates with over 90% visual motility and more 2×

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108 sperm/mL were mixed to avoid individual differences for cryopreserving in this

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study.

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Semen extension and freezing

Tris-citric-based extender (TCG) was the same with our previous study [33]. The

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freezing extenders were TCG with egg yolk (20%, v/v), DMSO (4%, v/v) and

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different concentrations of trehalose (0, 50, 75, 100,150, 200 mM).

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Semen samples were divided into six parts, diluted with the extender containing different concentrations of trehalose at 37 oC, and cooled to 5 oC. After that, the

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cooled semen was mixed with the freezing extenders and equilibration at 5 oC for 30

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min. Then, diluted semen samples were packed and sealed in 0.25 mL- straws

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immediately. Sperm were frozen and thawed as described by Zhu et al.(2015)[3].

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Sperm motility

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As described with Zhu et al.(2015) [33], 10 µL of post-thawed semen was placed

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on a pre-warmed clean glass slide. Sperm sample was evaluated under a

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phase-contrast microscope (Olympus, Japan) at 400× magnification. After viewing

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five different fields of the sample, sperm motility were estimated and noted by one

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observer (ZZD).

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Sperm membrane integrity

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Membrane integrity were assessed by LIVE/DEAD Sperm Viability Kit (Leiden

ACCEPTED MANUSCRIPT the Netherlands, L7011) [33]. Briefly, sperm supernatant were stained with SYBR-14

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(100 µM in DMSO) and propidium iodide (PI) (2.4mM in water) at 36 oC in the dark.

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Then, an epifluoresence microscope (Nikon 80i; Tokyo, Japan) was used to monitor

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and photograph the stained sperm in a set of filters (400×). 200 sperm were counted,

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and the treatments were replicated 5 times. All samples were evaluated by one

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observer (ZZD).

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Acrosome integrity

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Acrosome integrity was assessed by fluorescein isothiocyanate-peanut agglutinin

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(FITC-PNA, Sigma) solution (100 µg/mL) stained [33]. Briefly, sperm samples were

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fixed with absolute methanol for 10 min, and incubated with FITC-PNA solution (100

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µg/mL) in dark at 37℃for 30 min. Subsequently, samples were rinsed with PBS for

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three times prior to air-dried in dark. An epifluoresence microscope (Nikon 80i;

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Tokyo, Japan) with a set of filters (400X) were used to assess samples immediately.

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Photographs were also taken with a phase-contrast microscope in the same field, 200

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sperm were counted, and the treatments were replicated 5 times. All samples were

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evaluated by one observer (ZZD).

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Mitochondrial membrane potentials (ψm)

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JC-1 (lipophilic cation 5,5, ,6,6,- tetrachloro-1,1,,3,3,

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-tetraethylbenzimidazolcarbocyanine iodide) Mitochondrial Membrane Potential

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Detection Kit (Beyotime Institute of Biotechnology, Haimen, China) was used to

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analyze the changes of sperm mitochondrial membrane potential (∆Ψm) [9]. There

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are two types of JC-1 in stained mitochondrial plasma, one is a monomer, which emits

ACCEPTED MANUSCRIPT green fluorescence in a low mitochondrial membrane potential, and the other is an

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aggregates, which emits red fluorescence in a high mitochondrial membrane potential.

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Briefly, sperm samples (2×106/mL) were stained with JC-1 working solution (5 µM)

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at 37℃ for 30 min in the dark, centrifuged (600 x g, 5min) and washed with JC-1

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buffer and placed on ice. The stained samples were immediately estimated under a

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fluorescence microscope (Nikon 80i; Tokyo, Japan) with a set of filters (400X). High

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membrane potential was associated with emission at red (590 nm), low membrane

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potential was green (at 530 nm). At least 200 of spermatozoa were counted in each

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field. The treatments were replicated 5 times. All samples were evaluated by one

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observer (ZZD).

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Intracellular ROS measurement

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7′-dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime Institute of

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Biotechnology, Nanjing, China), according to Zhu et al. (2015) [33], intracellular

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DCFH-DA was deesterified to dichlorodihydrofluorescein which is oxidized by ROS

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to produce the fluorescent compound dichlorofluorescein. Sperm suspensions (10×106

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cells /mL) were incubated with 10 µM DCFH-DA at 37 °C for 30 min in the dark. The

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fluorescence intensity was measured using the fluorescence plate reader (Synergy HT,

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BioTek, USA) at Ex./Em. = 485/535 nm. The treatments were replicated 5 times.

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Intracellular MDA measurement

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Malondialdehyde (MDA) content was quantified by a commercial kit according to the manufacturer’s protocol [26]. In brief, sperm cellular extracts were prepared by

ACCEPTED MANUSCRIPT sonication (20KHz, 750W, operating at 40%, on 3s, off 5s, 5 cycles) in ice-cold buffer

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(50 mM Tris–HCl, pH 7.5, 5 mM EDTA, and 1 mM DTT), lysed cells were

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centrifuged at 12,000 × g for 10 min to remove debris after sonication. The

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supernatant was subjected to the measurement of MDA (at 532nm) levels with a

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microplate reader. MDA levels were then normalized to milligram protein.

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T-AOC activity

T-AOC activity was measured by using a T-AOC assay kit (Nanjing Jiancheng

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Bioengineering Institute, Nanjing, China). According to the manufacturer’s

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instructions, sperm samples were washed three times with TCG, then sperm pellets

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were re-suspended, lysed ultrasonically (20KHz, 750W, operating at 40%, on 3s, off

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5s, 5 cycles) on ice, then centrifuged at 12 000 ×g for 10 min at 4 °C, supernatants

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were mixed with the reaction buffer, then measured at 520 nm a microplate reader.

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Measurement of CAT and SOD activities The catalase assay kit and the total superoxide dismutase assay kit with NBT

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were used to assess the catalase (CAT) and superoxide dismutase (SOD) activities,

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respectively (Beyotime Institute of Biotechnology, Shanghai, China). Sperm pellets

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were rinsed three times with PBS buffer and re-suspended, lysed ultrasonically

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(20KHz, 750W, operating at 40%, on 3s, off 5s, 5 cycles) on ice, then centrifuged at

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12 000 ×g for 10 min at 4 °C. The supernatants were used to analyze the catalase

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(CAT) and superoxide dismutase (SOD) activities according to the manufacturer’s

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instructions.

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Statistical analysis Percentage data were transformed by arc-sin square root transformation to normalize distributions prior to statistical analysis. All data were analyzed by one-way

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ANOVA, and multiple comparisons with Tukey test was performed using SPSS

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version 17.0 for Windows (SPSS Inc., Chicago, IL). All the values are presented as

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mean ± standard deviation of the mean (SEM). A probability (p) value of less than

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0.05 was considered to be statistically significant.

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Result

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Sperm motility

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The value of sperm motility in the trehalose treatments was higher than that of

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control (Fig.1A). Especially, addition of 100 mM trehalose showed the highest

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motility among the treatments. These observations indicated that supplemented with

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trehalose could improve the post-thawed motility.

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Acrosome integrity

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Sperm stained with FITC-PNA were classified into 3 groups ( Fig. 2B): (a)

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damaged acrosome, (b) intact acrosome, (c) partially damaged acrosome. The effect of addition of different concentrations of trehalose on acrosome

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integrity was shown in Fig. 1B. Freezing extender supplemented with 100 mM

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trehalose showed a significantly higher value of acrosomal integrity than that of

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control. The integrity of acrosome in treatments between addition of 75 mM and 150

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mM trehalose were similar, and higher than the control. These data suggested that

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addition of trehalose protected the acrosome from damage during freezing-thawing

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process.

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Membrane integrity Membrane integrity was assessed by SYBY-14/PI staining. The spermatozoa

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were classified into three groups (Fig. 2A): sperm presented red by stained with PI but

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unstained with SYBR-14 (damaged membrane), sperm presented green-orange by

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stained both SYBR-14 and PI (membrane damaged slightly), sperm presented green

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by stained with SYBR-14 but unstained with PI (membrane integrity).

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In terms of membrane integrity, addition of 100 mM trehalose showed higher

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value than that of control (p <0.05). However, treatments addition of 150 mM or 200

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mM trehalose to the freezing extenders did not yield better results (Fig. 1C). These

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data suggested that the optimal concentration of trehalose added to the freezing

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extender was 100 mM trehalose.

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Mitochondria membrane potentials When the spermatozoa were incubated with fluorescent probe JC-1, two kinds of

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spermatozoa in mid-piece stained by probe were observed under a fluorescent

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microscope ( Fig. 3B). The yellow (orange) fluorescent indicated the spermatozoa

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were in high mitochondrial membrane potentials, while the green fluorescent

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indicated the spermatozoa were in low mitochondrial membrane potentials.

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Addition of trehalose significantly increased the value of mitochondrial

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membrane potentials (Fig. 1D). Compared with the control, the treatment with 100

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mM trehalose show maximum mitochondrial membrane potentials value (27.9% vs

ACCEPTED MANUSCRIPT 52.5%). Meanwhile, the percentage of mitochondrial membrane potentials of other

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three treatments were 44.8%, 44.7% and 35.9% (75, 150, 200 mM, respectively), but

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still were higher than that of control. The data indicated that supplementation of

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trehalose results in maintaining the mitochondrial membrane potentials.

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ROS generation and MDA production

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To elucidate whether addition of trehalose improved the parameters of

frozen-thawed spermatozoa by its antioxidantive ability, spermatozoa ROS and MDA

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content were measured. As shown in Fig. 3D, the high green fluorescence level

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presented that the spermatozoa were in high level of intracellular ROS, whereas the

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low green fluorescence indicated spermatozoa with low intracellular ROS. When

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compared with control, addition of trehalose significantly decreased spermatozoa

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ROS level (p< 0.05). The ROS level of post-thawed spermatozoa in the treatment

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supplemented with 100 mM of trehalose showed a significantly lower value than that

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of control (57.8% vs 100%) (Fig. 4A). The ROS level of post-thawed spermatozoa

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was decreased firstly and then increased with different concentrations of trehalose.

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We also measured the MDA content of post-thawed spermatozoa (Fig. 4B).The

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MDA content in the treatment of 100 mM trehalose were significantly lower than the

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control group (5.65 vs 12.32 nmol/mg protein). However, the MDA level of

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post-thawed spermatozoa were similar among the treatments supplemented with 50

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mM,75 mM and 200 mM of trehalose, but significantly lower than that of control.

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These observation indicated that addition of trehaolse in the freezing extender could

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decrease the post-thawed spermatozoa ROS level and MDA content to protect

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spermatozoa.

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Catalase activity

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Effects of different concentrations of trehalose on catalase activity of post-thaw spermatozoa were shown in Fig. 4C. Addition of 100 mM trehalose significantly

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improved the catalase activity of post-thawed sperm, when compared to the control

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(p< 0.05). However, the catalase activity in the treatment supplemented with 200 mM

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trehalose did not show a better improving result.

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Superoxide dismutase (SOD) activity and T-AOC activity

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In terms of superoxide dismutase (SOD) activity of post-thaw spermatozoa, supplementation of trehalose significantly improved superoxide dismutase (SOD)

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activities (p<0.05), except for the concentration of 50 mM trehalose. The treatments

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with 75 mM or 100 mM trehalose showed highest value among the treatments (Fig.

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4D). As shown in in Fig. 5, T-AOC activity of treatments supplemented with trehalose

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were higher than that of control (p< 0.05). Moreover, treatments addition of 100 or

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150 mM trehalose showed the highest value of T-AOC activity among all treatments.

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Those results indicated that supplementation of trehalose improved the activity of

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SOD and T-AOC.

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Discussion

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The present study was to investigate whether addition of trehalose to freezing

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extenders could protect sperm from cryo-damage in rabbits. It was found that freezing

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extenders added with 100 mM trehalose significantly improved the post-thawed

ACCEPTED MANUSCRIPT sperm parameters. Addition of 100 mM trehalose enhanced the activities of catalase

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and superoxide dismutase, decreased the ROS level and MDA content of post-thaw

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spermatozoa, suggested that trehalose protect post-thaw spermatozoa from

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cryodamage via its antioxidantive capacity.

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ROS was accumulated during sperm cryopreservation and incubation process in our previous study [33]. Excessively ROS was damaged to sperm via disrupting the

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balance between ROS production and detoxification by antioxidants, led to oxidative

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stress. When high levels of ROS accumulated in sperm, ROS resulted in LPO, caused

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loss of membrane integrity and fluidity. Moreover, this can lead to the motility

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declined rapidly and irreversibly.

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Long-chain polyunsaturated fatty acids (PUFA) were predominant in sperm membrane [5]. The unstable molecules (PUFA) make the sperm membrane

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susceptibly to peroxidation. Though the antioxidant defense systems of sperm and

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seminal plasma protect ejaculated freshly semen against oxidative stress [7,29], the

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capability of antioxidant is restricted for the dilution of semen. Furthermore, most of

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the cytoplasmic components are disposed during maturation stage of spermatogenesis,

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which resulted in decreasing the ability of antioxidants to counteract the harmful of

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ROS. Consequently, spermatozoa are deficient of natural antioxidants and sensitive to

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oxidative stress during the process of preservation, especially in cryopreservation.

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Therefore, addition of antioxidants can protect sperm against the detrimental effects

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of ROS and LPO, and improve the post-thaw sperm motility, membrane integrity and

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acrosome integrity.

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ACCEPTED MANUSCRIPT Trehalose, a non-reducing disaccharide sugar, is believed to have antioxidative

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property and protects sperm from ROS damaged [31]. The present study is consistent

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with Iqbal et al.(2016) [19], we found that supplementation of trehalose to the

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freezing extender significantly enhanced the activities of catalase and superoxide

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dismutase(Fig. 4C-D). Moreover, in our study, ROS production and MDA content of

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the post-thaw sperm were decreased with addition of trehalose (Fig. 4A-B), which

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was agreement with Chhillar et al.(2012) [10] and Aisen et al. (2005) [2]. Shiva

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Shankar Reddy et al.( 2010) [28] demonstrated that supplementation of trehalose

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elevated the T-AOC activity of post-thaw sperm, which is similar to the result in

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present study(Fig. 5). Those conformed that addition of trehalose to the extenders

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enhanced the antioxidant capability of the post-thaw sperm, decreased ROS

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accumulation, and resulted in preventing sperm from LPO to protect sperm. On the

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other hand, sperm fertilization potential is strongly related to ∆Ψm and thus to

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mitochondrial functionality [17,21]. Compared to the control, the high mitochondrial

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membrane potentials percentage of post-thaw sperm was significantly elevated with

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addition of trehalose, which was consistent with Najafi et al. (2013) [24] and

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Athurupana et al. (2015) [6]. As ∆Ψm was negatively correlate with the generation of

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ROS [32], addition of trehalose improved the ∆Ψm of post-thaw sperm by scavenging

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ROS. Therefore, supplementation of trehalose caused a significantly increase in the

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mitochondrial membrane potential of post-thaw rabbit spermatozoa.

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In terms of evaluation of sperm functionality, plasma membrane integrity and acrosome integrity are the main parameters [12]. In present study, the percentage of

ACCEPTED MANUSCRIPT membrane integrity of post-thaw spermatozoa treated with trehalose showed a higher

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value than that of control, which was in agreement with Aisen et al. [2] and Kumar

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and Atreja [19]. On the other hand, oxidative stress, thermal stress, osmotic stress or

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ice crystal formation caused acrosome damaged during cryopreservation [15].

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Addition of trehalose to the freezing extenders resulted in a significant increase of

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acrosome integrity as well, which was consistent with the results in boars[6, 14, 22],

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rams [3], goats [3]. These observations indicate that addition of trehalose to the

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freezing extenders provides greater structural integrity for rabbit spermatozoa after

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cryopreservation.

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In present study, motility of post-thaw rabbit sperm was significantly improved by

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addition of 100 mM or 150 mM trehalose. it is in agreement with the previous reports

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in rams [4], boars [6] and buffalos[19], which demonstrated that supplementation of

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trehaolse with the cryopreservation media prior to cryopreservation significantly

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enhanced survival rate of post-thaw spermatozoa. In conclusion, supplementation of trehalose results in higher activity of CAT and

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SOD, less ROS production and MDA content, and higher integrity of plasma

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membrane, acrosome and mitochondrial as well as higher motility for post-thaw

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rabbit spermatozoa. Addition of trehalose prior to cooling process may be

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recommended to facilitate the improvement of semen preservation technique for the

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rabbit breeding industry.

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Acknowledgement This study was supported in part by the National Natural Science Foundation of China (Grant No. 31072029, No.31272439 and No. 31230048) for W Zeng.

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Competing Interests

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The authors declared that they had no competing financial interests.

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Figure Legends

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Fig. 1. Effect of trehalose on post-thaw sperm motility (A), acrosome integrity (B),

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membrane integrity (C) and mitochondrial membrane potentials (D). Bars represent

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the mean ± SEM (n = 5 independent replicates). Different lower-case letters denote

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significant differences (p < 0.05).

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Fig. 2. Photomicrographs of the post-thaw rabbit spermatozoa. Images (C, D)

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obtained under phase contrast microscope. Images (A) and (C) were from the same

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field, blue arrows indicates membrane integrity; black arrows indicates membrane

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damaged, white arrows indicates membrane damaged slightly. Images (B) and (D)

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were from the same field. Red arrow indicates damaged acrosome; blue arrows

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indicates intact acrosomes; white arrow indicates partially damaged acrosome. Bars =

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24 µm.

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Fig. 3. Photomicrographs of the post-thaw rabbit spermatozoa. Images (A) and (B)

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were from the same field. Blue arrows indicate sperm with high mitochondrial

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membrane potentials, red arrow indicates sperm with high mitochondrial membrane

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potentials. Images (C) and (D) were from the same field. white arrow indicate sperm

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with higher level of ROS, while yellow arrow indicates sperm with lower ROS. Bars

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= 30 µm.

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activity (C) and Superoxide dismutase activity(D) of post-thaw rabbit sperm. Bars

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represent the mean ± SEM (n = 5 independent replicates). Different lower-case letters

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denote significant differences (p < 0.05).

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Fig. 5. Effect of trehalose on T-AOC activity of post-thaw spermatozoa. Bars

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represent the mean ± SEM (n = 5 independent replicates). Different lower-case letters

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denote significant differences (p < 0.05).

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