- Email: [email protected]
Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation
Cryobiology xxx (2016) 1e5
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation Napapach Chuaychu-noo a, Pachara Thananurak a, Vibantita Chankitisakul a, Thevin Vongpralub a, b, * a b
Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand Research and Development Network Center for Animal Breeding (Native Chicken), Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 June 2016 Received in revised form 6 November 2016 Accepted 29 December 2016 Available online xxx
Little is known about the effects of Cholesterol-Loaded Cyclodextrin (CLC) on post-thaw semen quality in chicken. The aim of the present study is to investigate the efficacy of CLC levels (0, 1, 2 and 3 mg/mL Schramm diluent) on post-thawed semen quality and fertility in two breeds of chicken Pradu Hang Dum (native chicken) and Rhode Island Red. Semen samples of each breed were pooled, divided into 4 aliquots and diluted with Schramm diluents, cooled to 5 C when DMF was added (6% of final volume). Semen straws were subjected to cryopreservation using the liquid nitrogen vapor method. Post-thawed sperm motility, viability, acrosome integrity, mitochondrial function, and the Malondialdehyde (MDA) level were determined. The fertility of frozen semen was tested by inseminating laying hens. Post-thaw motility between Pradu Hang Dum and Rhode Island Red was no different; but Rhode Island Red had a higher semen viability and live cell intact acrosomes than Pradu Hang Dum (P < 0.05). The percentage of high functioning mitochondria in the Pradu Hang Dum was higher than the Rhode Island Red. CLC at 2 and 3 mg/mL supplementation was associated with improved viability of frozen semen; that is, acrosome integrity and mitochondrial function (P < 0.01), albeit having no effect on MDA levels. The sperm with 1 mg/mL CLC yielded a significantly better fertility (P < 0.01). CLC (1 mg/mL) improved the quality of frozen rooster semen. There was no interaction among breeds and CLC on post-thaw semen quality and fertility. © 2016 Published by Elsevier Inc.
Keywords: Breeds Rooster Cholesterol Load Cyclodextrin (CLC) Frozen semen Fertility
1. Introduction Semen cryopreservation is a crucial biotechnological method of ex situ conservation for preventing genetic loss. The application of this technique for cattle has grown rapidly but sperm freezing for poultry is limited because of the high costs of semen processing and subsequent lower fertility [3]. Membrane damage during cooling is a major cause for sperm death during cryo-preservation [21]. Membrane lipid composition plays a significant role in a sperm's response to cooling and warming during cryopreservation [1,6]. Sperm membrane comprises of lipids, cholesterol, and proteins in specific arrangements, and the cholesterol/phospholipid ratio plays a major role in membrane fluidity [8]. Membranes with high cholesterol to
* Corresponding author. Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand. E-mail address: [email protected] (T. Vongpralub).
phospholipid ratios exhibit lower transition temperatures, which allow the membrane to remain fluid through most of the freezing process and this decreases the amount of membrane damage during cooling and warming [34,23]. Those ratios in roosters are, however, quite low (0.25e0.30) [4,23]. Cyclodextrins are cyclic oligosaccharides formed from starch through cyclodextrin glycosyltransferase action [11]. Cyclodextrins have a hydrophilic surface and a hydrophobic core, which allows the molecules to be soluble in aqueous solutions but allows hydrophobic molecules to solubilize in cyclodextrin. Hydrophobic molecules in cyclodextrin can be solubilized in an aqueous solution, without structural change [7,10]. Pre-loaded Cholesterol (CLC) molecules have been used to incorporate cholesterol in to the sperm membranes and increase the cholesterol content of sperm membranes [20,29]. When the cholesterol content of stallion, boar, ram, and bull sperm is increased by CLC, the cryosurvival rate and quality of the frozen-thawed sperm is increased [2,15,19,20]. This decreases the membrane damage upon warming [5]; therefore, CLC
http://dx.doi.org/10.1016/j.cryobiol.2016.12.012 0011-2240/© 2016 Published by Elsevier Inc.
Please cite this article in press as: N. Chuaychu-noo, et al., Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.12.012
2
N. Chuaychu-noo et al. / Cryobiology xxx (2016) 1e5
Unless otherwise stated, all chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). The cell viability test kit was obtained from the Life Technologies (Carl.sbad, CA, USA).
(O.P.) ¼ 395 mOsm/kg) [31]. Immediately after collection of the semen sample from each breed group, these were pooled separately in 15 mL tubes. Semen in each tube was divided into 4 aliquots and diluted (to 0, 1, 2 and 3 mg/1 mL) of CLC, Schramm-based diluter 1: 3 (v: v) was then cooled to 5 C for 60 min (1 C per 3 min). At this temperature, the DMF (N,N-dimethylformamide) was added to a final concentration of 6% (v/v) and then mixed. Semen was immediately loaded into 0.5 mL plastic straws and sealed with Polyvinylpyrrolidone powder (PVP). The number of final sperm concentration in each straw was about 500 106. After 15 min of equilibration, the filled straws were laid horizontally on a rack 11 cm above the surface of liquid nitrogen (35 C) for 12 min then placed 3 cm above the liquid nitrogen vapor (135 C) for 5 min, and subsequently immersed in liquid nitrogen for storage at 196 C until analyzed. Thawing was achieved at 2-5 C for 5 min in cool water [33].
2.2. Animals
2.6. Evaluation sperm quality
Twelve Pradu Hang Dum (Thai Native) and twelve Rhode Island Red, 1 to 2-year-old breeder male roosters were housed in individual cages, fed 130 g/d/b of commercial feed, and given water ad libitum. Forty-eight individually housed, commercial laying hens (24 weeks of age) having egg production of 85e95% were fed 110 g/ d/b of layer feed and given water ad libitum. The animals were reared under natural environment conditions. The study was conducted at the research farm, Department of Animal Science, Faculty of Agriculture, Khon Kaen University; Thailand. The Animal Ethics Committee of Khon Kaen University reviewed and approved the study (Approval No.: 0514.1.12.2/29).
The concentration of sperm was determined with a haemocytometer under a light microscope. The computer-assisted sperm analysis system (CASA) (version 10 HIM-IVOS; Hamilton Thorne Biosciences, Beverly, MA USA) has the ability to assess total motility (MOT), progressive motility (PMOT), velocity average path (VAP), velocity straight line (VSL), and velocity curvilinear (VCL) [12]. The percentage viable was determined by SYBR - 14 and Propidiam Iodide (PI) (Live/Dead Sperm Viability Kit, InvitrogenTH, Thermo Fisher Scientific, Waltham, MA, USA) [24]. The percentage of acrosome integrity and functional mitochondria was determined by PI (Sigma, 81845), 5, 50 , 6, 6’ e tetraethybenzimdazolycarboncyanine iodide (JC- 1; Sigma, C50390) and fluorescein isothiocyanate conjugated peanut agglutinin (FITC-PNA; Sigma, L7381). These were evaluated by flow cytometry (FACSalibur; Becton Dickinson, San Jose, CA, USA) as described by LangeConsiglio [13] with minor modifications.
treatment prior to cryopreservation may be able to decrease rooster sperm damage after cryopreservation and increase fertility. To our knowledge, there is no published information on the effects of CLC in chicken cryopreserved semen. Our aim therefore was to determine whether treating rooster sperm with CLC prior to cryopreservation would improve post-thaw sperm survival and also to optimize the concentration of CLC suitable in the semen freezing diluent for improving fertility. 2. Materials and methods 2.1. Chemicals
2.3. Preparation of cyclodextrin Cholesterol-Loaded Cyclodextrin (CLC) was prepared as per Purdy and Graham [27]. Briefly, 1 g of cyclodextrin was dissolved in 2 mL of methanol in a glass tube. In a second glass tube, 200 mg of cholesterol was dissolved in 1 mL of chloroform. The cyclodextrin/ methanol solution was added to the cholesterol/chloroform solution and mixed (glass tube 1 into 2). The solution (in a petridish) was then subjected to a stream of nitrogen gas to vapor at the solvent and to obtain a white CLC, which was stored at 5 C. The CLC working solution was mixed with Schramm diluent, using a vortex mixer for 30 s [27]. 2.4. Semen collection Semen was collected twice a week, by the dorso-abdominal massage method [28]. Semen from an individual rooster was collected in a 1.5 mL micro-tube containing 0.1 mL of Schramm diluent. To maximize semen quality and quantity, the collection was performed by the same person, under the same conditions, at the same time, and using the massage method. Care was taken to avoid contamination of semen with feces, urates, and transparent fluid-all of which lowers the semen quality. Semen samples were selected on the basis of meeting the following criteria: mass motility score 4; sperm concentration 3 109 sperm/mL; sperm viability 85%; and, 90% normal cells in the semen sample [33]. 2.5. Semen dilution and cryopreservation Schramm diluents was used in the current study, comprising of magnesium acetate 0.7 g, sodium glutamate 28.5 g, glucose 5 g, inositol 2.5 g, and potassium acetate 5 g, all of which were dissolved in 1,000mL of double-distilled water (pH ¼ 7.1, Osmotic pressure
2.7. Lipid peroxidation Malondialdhyde (MDA) concentration as indices of the lipid peroxidation in the semen samples was measured using the Thiobarbituric acid (TBA) reaction. Semen samples from each treatment 250 mL (250 106 spz/mL) was centrifuged at 1,800 rpm for 3 min and the supernatant discarded and 1 mL of Schramm diluent added. The semen pellet was added with 0.25 mL Ferrous sulfate (Ajex, 0906251) (0.2 mM) and 0.25 mL ascorbic acid (Sigma, A5960) (1 mM). The mixture was incubated at 37 C for 60 min after which 1 mL trichloroacetic acid (Sigma, T6399) (15%) and 1 mL thiobarbituric acid (Sigma, T550) (0.375%) were added, and boiled in water for 10 min. Thereafter, the samples were cooled to room temperature to stop the reaction. Finally, the samples were centrifuged to controlled temperature of 4 C at 5,000 rpm for 10 min and analyzed, using UVeVisible spectrophotometer (Analytikjena Model Specord 250 plus) at 532 nm [24]. 2.8. Fertility The fertility ability of frozen-thawed spermatozoa was tested by inseminating layer hens (48 hens were randomly assigned to 8 groups of 6 hens) once a week with a dose of 0.4 mL. All inseminations were performed between 15.00 and 17.00 Hrs. Eggs were collected during days 2e8 after insemination. Fertility was determined by candling eggs on day 7 of incubation. Six replications of fertility test were carried out. For the fertile period test, 48 hens (about 80% of hen-day production) were randomly assigned to 3 groups, and inseminated
Please cite this article in press as: N. Chuaychu-noo, et al., Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.12.012
N. Chuaychu-noo et al. / Cryobiology xxx (2016) 1e5
with fresh semen, frozen semen (control), and the best group from experiment of CLC supplemented were tested by inseminating layer hens once a week with a dose of 0.4 mL. All insemination was performed at 15.00e17.00 Hrs. Eggs were collected during days 2e16 after insemination. Fertility was determined by candling eggs on day 7 of incubation [33]. 2.9. Statistical analysis The experiments were conducted using 2 4 factorial randomized complete block design (RCBD). Statistical analysis was performed using the General Linear Model (GLM). The mean of the CLC treatment effects were compared using Duncan's Multiple Range test (DMRT) (P < 0.05). The arcsine square root transformation was used SAS [30]. Six replications were used for evaluating sperm quality with flow cytometry, CASA, MDA, fertility test, and duration of fertility respectively. 3. Results 3.1. Effects of CLC supplementation of diluent on sperm motility and sperm viability in different breeds of rooster The effects of breeds on frozen semen quality are presented in Table 1. Rhode Island Red had a higher sperm viability than Pradu Hang Dum (P < 0.05) but there was no difference in motility between breeds. CLC supplementation (2 and 3 mg/mL) of frozen semen resulted in a higher quality over against the without control (P < 0.01). Neither breed nor CLC supplementation had any appreciable effect on percentage of MOT, PMOT, VAP, VSL, VCL. Rhode Island Red had higher live cells with intact acrosomes (P < 0.05) than Pradu Hang Dum (Table 2). Supplementation with 1, 2 and 3 mg/mL CLC in the diluents resulted in a higher percentage of live with intact acrosome (P < 0.01) than the control group. Pradu Hang Dum with functioning mitochondria was greater (P < 0.01) than Rhode Island Red and CLC supplementation (1, 2 and 3 mg/mL) was greater than the control group (Table 2). 3.2. Effects of CLC supplementation of diluent on fertility and lipid peroxidation in semen The effects of CLC supplementation of Scharmm extender on fertility in different breeds are presented in Table 2. Breeds had no effect on fertility rate; however, supplementation of 1 mg/mL CLC in the freezing extender improved the fertility of frozen semen (P < 0.01). CLC had no effect on the MDA levels in frozen semen
3
(P > 0.05). 3.3. Effects of CLC supplementation of diluent on fertile period Fertility rates were compared after Artificial Insemination (AI) using fresh semen and frozen semen, control, and 1 mg/mL CLC supplementation in diluents for 3 periods post AI (days 1e7, 8e14 and 1e14) (Fig. 1). The results showed that the fertility rate of fresh semen was the highest and that frozen semen treated with CLC was higher than the control group. Frozen semen on days 1e7 was 88.85 ± 5.23, 81.63 ± 4.40 and 73.10 ± 6.63%; on days 8e14 was 48.43 ± 1.68, 23.30 ± 4.55 and 14.14 ± 3.67%; and on days 1e14 was 66.42 ± 1.17, 54.08 ± 2.45, and 42.26 ± 3.36% respectively. 4. Discussion The current study was under taken to obtain a suitably modified semen diluent for improving the quality of the cryopreserved -vis fertility rate after AI under field consemen of chickens' vis-a ditions. Based on the results, we concluded that supplementation with 1% CLC improves post-thaw semen quality in chickens. To our knowledge, this is the first report on the effects of CLC on frozen semen quality in chickens. Rooster sperm with CLC improved sperm viability and membrane integrity of the sperm cells in a manner similar to CLC supplementation of mammalian sperms [23]. The results of the experiment show that CLC at 1, 2 and 3 mg/ mL improved rooster frozen semen quality. This is similar to the optimal CLC concentration reported for improving the quality of the frozen semen of bulls, stallions, boars, and goats [19,20,27,29,32]. Previous reports demonstrated that semen diluent supplemented with CLC improved sperm survival, motility, membrane integrity, and fertility in many farm animal species [18]. The cholesterol to phospholipid ratio is an important determinant of cryosurvival rates and is different for sperms from different species, lines within a species, males, and after various treatments [19]. These components are crucial for sperm membrane fluidity and stability at low temperatures. Nevertheless, the benefit of CLC treatments modifies the capacitation pattern, timing of sperm, and subsequent ability of sperm to undergo the acrosome reaction. Although avian spermatozoa do not seem to undergo capacitation or motility hyper-activation prior to fertilization [16], they do spend a very long time in the female oviduct before oocyte penetration [9]. Howarth [9] reported that fowl sperm have been shown to undergo acrosome reaction; thus fertilizing the ovum directly without requiring capacitation. Possible in vivo fertilization in chickens, the perivitelline layer just after ovulation is composed of
Table 1 Sperm quality parameters of frozen semen of 2 breeds of rooster at 4 concentrations of CLC supplementation (mean ± SD; n ¼ 12). Factor Breeds
VI(%)
MOT(%)
PMOT(%)
VSL(mm/s)
VCL(mm/s)
VAP(mm/s)
Pradu Rhodes
43.63 ± 4.42 45.90 ± 3.98a
57.45 ± 7.80 60.90 ± 7.52
34.24 ± 3.57 36.50 ± 3.83
76.71 ± 6.31 77.88 ± 6.04
131.90 ± 6.13 134.35 ± 6.63
91.45 ± 5.55 92.94 ± 7.89
0.03
0.21
0.08
0.53
0.23
0.50
0 1 2 3
40.60 42.62 44.67 45.04
P- value Level of CLC (mg/mL)
b
± ± ± ±
2.47C 2.91B 3.16A 4.78A
58.60 58.20 59.90 60.00
± ± ± ±
5.51 9.65 8.17 6.81
34.03 34.83 36.03 36.60
± ± ± ±
3.03 4.21 3.07 4.57
75.23 79.58 77.66 76.72
± ± ± ±
3.52 7.36 5.81 7.05
133.23 133.96 132.96 128.37
± ± ± ±
2.77 7.87 5.24 9.00
90.64 92.55 92.78 92.82
P- value
0.001
0.95
0.48
0.43
0.95
0.88
Interaction
0.57
0.68
0.39
0.59
0.50
0.62
± ± ± ±
3.36 6.75 7.27 9.75
Different letters (A,B) within columns indicate significant differences (P < 0.01). Different letters (a,b) within columns indicate significant differences (P < 0.05), VI (%): percentage of viable spermatozoa with an intact plasma membrane assessed by propidium iodide staining (PI negative); MOT (%): percentage of motile spermatozoa; PMOT (%): percentage of spermatozoa with progressive motility; VAP (mm/s): percentage of velocity average path; VSL (mm/s): percentage of velocity straight line; VCL (mm/s) percentage of velocity curvilinear.
Please cite this article in press as: N. Chuaychu-noo, et al., Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.12.012
4
N. Chuaychu-noo et al. / Cryobiology xxx (2016) 1e5
Table 2 Effects of CLC supplementation of the diluent on acrosome integrity, functional mitochondria on fertility, and malondiodehyde of rooster spermatozoa in frozen-thawed semen (mean ± SD; n ¼ 12). Live with intact acrosomes
Functional mitochondria
MDA (mm/mL/250 106spz)
% Fertility (egg set)
Pradu Rhode
27.32 ± 4.37b 29.15 ± 6.88a
38.28 ± 4.58A 36.30 ± 6.75B
1.67 ± 0.53 1.53 ± 0.33
85.73 ± 9.38 (995) 87.61 ± 7.84 (913)
0.04
0.005
0.27
0.31
0 1 2 3
20.03 29.56 30.56 31.80
Factor Breed P- value Level of CLC (mg/ml)
± ± ± ±
2.62B 1.80A 2.50A 4.54A
29.17 37.28 39.96 42.55
± ± ± ±
3.20C 3.19B 1.82AB 3.00A
1.51 1.46 1.77 1.67
± ± ± ±
0.47 0.25 0.59 0.37
85.90 91.90 84.60 82.29
P-value
0.001
0.001
0.95
0.003
AxB
0.06
0.06
0.42
0.09
± ± ± ±
7.98B (448) 4.31A (492) 9.63B (481) 9.38B (447)
Different letters (A,B) within columns indicate significant differences (P < 0.01), Different letters (a,b) within columns indicate significant differences (P < 0.05), MDA; malondiodehyde.
Fig. 1. Fertile period after one artificial insemination. Different letters (A, B) with in bar indicate significant difference (p < 0.01).
inner perivitelline membrane, which is secreted just after fertilization in the upper part of oviduct. As studied in vitro, the results demonstrated that the acrosome reaction may be induced within a minute [14]. According to in vitro experiments, in mammalian species CLC extended sperm viability and acrosome integrity both in fresh and cryopreserved forms [19]. Our result indicates that sperm motility, sperm viability and sperm viability with heightened mitochondrial function are higher in spermatozoa treated with CLC than the untreated one and this parameter was higher in those treated with 2 and 3 mg CLC than those treated with 1 mg CLC. Recently, a study of the effect of CLC on sperm viability and acrosome reaction in live semen cryopreservation showed higher cholesterol incorporation in the acrosome membrane than the controlled. CLC not only reduced the premature acrosome reaction but also protected the viability of sperm and prevented premature acrosome reaction and capacitation, usually a side effect of cryodamage [15]. The in vitro assessment of sperm characteristic is important for determining sperm fertility, but those parameters are not conclusive. Thus, a fertility trial is needed to elucidate the real effect on fertility. The reason as to why the increased sperm quality does not
translate into a higher fertilizing ability is not known [18]. This may be due to CLC-treated sperm requiring more time to capacitate in the female reproductive tract, resulting in appropriate timing of AI i.e., between the acrosome reaction of the sperm and ovulation [19]. We hypothesize that in the current study, the low fertility rate of 2 and 3 mg CLC groups may be due to acrosome reactionpreventive property of CLC, which may have interfered with the sperm's penetrating ability through the inner perivitelline membrane. Thus, supplementation of CLC (more than 1 mg/mL) may interfere with the acrosome reaction leading to a decline in fertility rates. Semen supplemented with the optimal CLC concentration thus benefitted sperm characteristics, including sperm membrane integrity, viability, and fertilizing potential. The current study was performed to determine the effects of CLC on two breeds of chicken namely, Pradu Hang Dum and Rhode Island Red. The results showed that there were differences between -vis in percentage sperm viability i.e., live with the breeds vis-a intact acrosome: the Rhode Island Red had greater viability than Pradu Hang Dum. Pradu Hang Dum had more live with high mitochondrial activity than the Rhode Island Red. There was, however, no difference between breeds on post-thawed semen
Please cite this article in press as: N. Chuaychu-noo, et al., Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.12.012
N. Chuaychu-noo et al. / Cryobiology xxx (2016) 1e5
fertility. The success of freezing poultry semen is highly variable and depends on the species and the specific breed lines [3,17,22,25,35]. The percentage of sperm viability that is live with intact acrosome and live with high mitochondrial activity is correlated to fertility [3]. The number of surviving sperm in the reproductive tract may not be different between these two breeds, but the fertilizing ability of the frozen sperm of both breeds had similar fertilization rates. Although the fertility and fertile period of fresh semen is better than the frozen semen groups, the fertility rate and fertility period of the CLC dsupplemented group was superior to the frozen semen control group (Fig. 1). The duration of fertility was correlated with the number of sperm in the SST of the UVJ [26]. The results suggest that supplementation with 1 mg/mL CLC in the freezing diluents would improve survival and longevity of sperms over against the control frozen semen. In conclusion, CLC in freezing diluent improves the quality of frozen semen and subsequent fertility rates over against nonfrozen sperm. In the current study, there was no difference among breeds of rooster on post-thaw fertility. Taken together, the results of this study suggest that 1 mg/mL of CLC in the freezing diluent is an optimal concentration for improving the subsequent fertility of chickens impregnated with the thawed frozen sperms. Statement of funding Experiment was designed by T. Vongpralub, N. Chuaychu-noo. Semen freezing and post-thawed semen evaluations were done by N. Chuaychu-noo and P. Thananurak. Artificial insemination was carried on by T. Vongpralub, N. Chuaychu-noo and P. Thananurak. Manuscript was written by T. Vongpralub and V. Chankitisakul. Acknowledgements The authors thank (1) the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Food and Functional Food Research Cluster of Khon Kaen University (URU591005) for financial support; (2) the Research and Development Network Center for Animal Breeding (Native Chicken), Department of Animal Science, Faculty of Agriculture, Khon Kaen University (3) Institute of Agricultural Technology, Suranaree University of Technology for semen quality evaluation equipments. (4) Rajamangala University of Technology Srivijaya was supported by a PhD grant. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cryobiol.2016.12.012 References [1] R.P. Amann, J.K. Graham, Spermatozoal function, in: V.O. McKinnon, J.L. Voss (Eds.), Equine Reproduction, Lea and Febiger, Philadelphia, London, 1993, pp. 715e745. [2] E.A.M. Amorim, J.K. Gramham, B. Spirizzi, M. Meyers, C.A.A. Torres, Effect of cholesterol or cholesteryl conjugates on the cryosurvival of bull sperm, Cryobiology 58 (2009) 210e214. [3] E. Blesbois, F. Seigneurin, I. Grasseau, C. Limouzin, J. Besnard, D. Gourichon, G. Coquerella, P. Rauit, M. Tixier-Boichard, Semen cryopreservation for ex-situ management of genetic diversity in chickens creation of the French Avian Cryobank, Poult. Sci. 86 (2007) 555e564. [4] E. Blesbois, U.I. Grassea, F. Seigneurin, Membrane fluidity and the ability of domestic bird spermatozoa to survive cryopreservation, Reprod 129 (2005) 371e378. [5] D.C. Bongalhardo, S. Leeson, M.M. Buhr, Dietary lipids differentially affect membranes from different areas of rooster sperm, Poult. Sci. 88 (2009) 1060e1069.
5
[6] C.A. Castellano, I. Audet, J.L. Bailey, J.P. Laforest, J.J. Matte, Dietary omega-3 fatty acids (fish oils) have limited effects on boar semen stored at 17 C or Cryopreserved, Theriogenology 74 (2010) 1482e1490. [7] H. Dobziuk, Molecules with holes e cyclodextrins, in: H. Dodziuk (Ed.), Cyclodextrins and Their Complexes, Wiley-VCH VerlagGmbH&Co, Weinheim, Germany, 2006. [8] R.H. Hammerstedt, J.K. Graham, Cryopreservation of poultry sperm: the enigma of glycerol, Cryobiology 29 (1992) 26e38. [9] J.R. Howarth, An examination for sperm capacitation in the fowl, Biol. Reprod. 3 (1971) 338e341. [10] J. Hu, Z. Tao, S. Li, B. Liu, Review: the effect of cyclodextrins on polymer preparation, J. Mater. Sci. 40 (2005) 6057e6061. [11] Z.E. Jouni, J. Zamora, M. Synder, W.R. Montfort, A. Weichsel, M.A. Wells, aCyclodextrin extracts diacylglycerol from insect high density lipoproteins, J. Lipid Res. 41 (2000) 933e939. [12] L.M. King, R.H. Denise, A.M. Donoghue, Correlation of CASA velocity and linearity parameters with sperm mobility phenotype in turkeys, J. Androl. 21 (2000) 65e71. [13] A. Lange-Consiglio, A. Meucci, F. Cremonesi, Fluorescent multiple staining and CASA system to assess boar sperm viability and membranes integrity in short and long-term exten, Open Veterinary J. 3 (1) (2013) 21e35. [14] M. Lemoine, I. Grasseau, J.P. Brillard, E. Blesbois, A reappraisal of the factors involved in in vitro initiation of the acrosome reaction in chicken spermatozoa, Reprod 136 (2008) 391e399. [15] Y.S. Lee, S. Lee, S.H. Lee, B.K. Yang, C.K. Park, Effect of cholesterol-loadedcyclodextrin on sperm viability and acrosome reaction in boar semen cryopreservation, Anim. Reprod. Sci. (2015) 124e130. [16] M. Lemoinea, S. Mignon-Grasteaue, I. Grasseaua, M. Magistrinia, E. Blesboisa, Ability of chicken spermatozoa to undergo acrosome reaction after liquid storage or Cryopreservation, Theriogenology 75 (2011) 122e130. [17] A. Massip, S.P. Leibo, E. Blesbos, Cryobiology of gametes and the breeding of domestic animals, in: E. Benson, B. Fuller, N. Lane (Eds.), Live in the Frozen State, Taylor & Francis Group, London, 2004, pp. 371e393. , E. Blanch, C. Toma s, J.K. Graham, Use of cholesterol in sperm cryo[18] E. Moce preservation: present moment and perspective to future, Reprod. Domest. Anim. 45 (2010) 57e66. [19] E. fluorescein isothiocyanate conjugated peanut agglutinin (FITC-PNA; Sigma, L7381). These were evaluated by flow cytometry P.H. Purdy, J.K. Graham, Treating ram sperm with cholesterol-loaded cyclodextrins improves cryosurvival, Anim. Reprod. Sci. 118 (2010) 236e247. [20] A.I. Moore, E.L. Squires, J.K. Graham, Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival, Cryobiology 51 (2005) 241e249. [21] G.J. Morris, E. Acton, B.J. Murray, F. Fonseca, Freezing injury: the special case of the sperm cell, Cryobiology 64 (2012) 71e80. [22] T.M.B. Mosenene, Characterization and Cryopreservation of Semen of Four South African Chicken Breeds. Magister Scientiae Agriculturae, Faculty of Natural in Agricultural Sciences, University of the Free State Bloemfontein, Bloemfontein, 2009. [23] J.E. Parks, D.V. Lynch, Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes, Cryobiology 29 (1992) 255e266. [24] A. Partika, W. Nizanski, E. Lukaszewicz, Evalution of fresh and frozen-thawed fowl semen by flow cytometry, Theriogenology 74 (2010) 1019e1027. [25] S.O. Peters, O.D. Shoyebo, B.M. Ilori, M.O. Ozoje, C.O.N. Ikeobi, O.A. Adebambo, Semen quality traits of seven strain of chickens raised in the humid tropics, Int. J. Poult. Sci. 7 (2008) 949e953. [26] E.E. Pierson, G.R. McDaniel, L.M. Krista, Relationship between fertility duration andin vivo sperm storage in broiler breeder hens, Brit Poult. Sci. 29 (1988) 199e203. [27] P.H. Purdy, J.K. Graham, Effect of adding cholesterol to bull sperm membranes on sperm capacitation, the acrosome reaction, and fertility, Biol. Reprod. 71 (2004) 522e527. [28] J.P. Quinn, W.H. Burrows, Artificial insemination of fowls, J. Hered. 27 (1937) 31e37. [29] B.E. Spizziri, M.H. Fox, J.E. Bruemmer, E.L. Squires, J.K. Graham, Cholesterolloaded cyclodextrins and fertility potential of stallions spermatozoa, Anim. Reprod. Sci. 118 (2010) 255e264. [30] SAS Institute Inc, SAS/STAT User's Guide: Version 6.12, fourth ed., SAS Institute Inc., Carry, North Carolina, 1966. [31] G.P. Schramm, Suitability of different antifreeze agents for cryoprotection of cock sperm, Monatsh. Fuer Veterinaermedizin 46 (1991) 438e440. z, E. Blanch, M. Hern [32] C. Toma andez, M.A. Gil, J. Roca, J.M. V azquez, , Treating boar sperm with cholesterol-loaded cycloE.A. Martínez, E. Moce dextrins widens the sperm osmotic tolerance limits and enhances the in vitro sperm fertilising ability, Anim. Reprod. Sci. 129 (2011) 209e220. [33] T. Vongpralub, A. Utha, J. Pongpeng, P. Sonseeda and W.Yindee, A comparison of simple vapour method and programmable freezer on motility and fertility of frozen Thai native chicken semen, in: The 3 rd International Conference on Sustainable Animal Agriculture for Developing Countries (SAADC 2001), July 26-29 2011, Nakhon Ratchasima, Thailand, 2011. [34] P.F. Watson, The causes of reduced fertility with cryopreserved semen, Anim. Reprod. Sci. 60e61 (2000) 481e491. [35] G.J. Wishart, Semen quality and semen storage, in: P.M. Hocking (Ed.), Biology of Breeding Poultry, CAB Int, 2009, pp. 151e178.
Please cite this article in press as: N. Chuaychu-noo, et al., Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.12.012
Contents lists available at ScienceDirect
Cryobiology journal homepage: www.elsevier.com/locate/ycryo
Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation Napapach Chuaychu-noo a, Pachara Thananurak a, Vibantita Chankitisakul a, Thevin Vongpralub a, b, * a b
Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand Research and Development Network Center for Animal Breeding (Native Chicken), Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 June 2016 Received in revised form 6 November 2016 Accepted 29 December 2016 Available online xxx
Little is known about the effects of Cholesterol-Loaded Cyclodextrin (CLC) on post-thaw semen quality in chicken. The aim of the present study is to investigate the efficacy of CLC levels (0, 1, 2 and 3 mg/mL Schramm diluent) on post-thawed semen quality and fertility in two breeds of chicken Pradu Hang Dum (native chicken) and Rhode Island Red. Semen samples of each breed were pooled, divided into 4 aliquots and diluted with Schramm diluents, cooled to 5 C when DMF was added (6% of final volume). Semen straws were subjected to cryopreservation using the liquid nitrogen vapor method. Post-thawed sperm motility, viability, acrosome integrity, mitochondrial function, and the Malondialdehyde (MDA) level were determined. The fertility of frozen semen was tested by inseminating laying hens. Post-thaw motility between Pradu Hang Dum and Rhode Island Red was no different; but Rhode Island Red had a higher semen viability and live cell intact acrosomes than Pradu Hang Dum (P < 0.05). The percentage of high functioning mitochondria in the Pradu Hang Dum was higher than the Rhode Island Red. CLC at 2 and 3 mg/mL supplementation was associated with improved viability of frozen semen; that is, acrosome integrity and mitochondrial function (P < 0.01), albeit having no effect on MDA levels. The sperm with 1 mg/mL CLC yielded a significantly better fertility (P < 0.01). CLC (1 mg/mL) improved the quality of frozen rooster semen. There was no interaction among breeds and CLC on post-thaw semen quality and fertility. © 2016 Published by Elsevier Inc.
Keywords: Breeds Rooster Cholesterol Load Cyclodextrin (CLC) Frozen semen Fertility
1. Introduction Semen cryopreservation is a crucial biotechnological method of ex situ conservation for preventing genetic loss. The application of this technique for cattle has grown rapidly but sperm freezing for poultry is limited because of the high costs of semen processing and subsequent lower fertility [3]. Membrane damage during cooling is a major cause for sperm death during cryo-preservation [21]. Membrane lipid composition plays a significant role in a sperm's response to cooling and warming during cryopreservation [1,6]. Sperm membrane comprises of lipids, cholesterol, and proteins in specific arrangements, and the cholesterol/phospholipid ratio plays a major role in membrane fluidity [8]. Membranes with high cholesterol to
* Corresponding author. Department of Animal Science, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand. E-mail address: [email protected] (T. Vongpralub).
phospholipid ratios exhibit lower transition temperatures, which allow the membrane to remain fluid through most of the freezing process and this decreases the amount of membrane damage during cooling and warming [34,23]. Those ratios in roosters are, however, quite low (0.25e0.30) [4,23]. Cyclodextrins are cyclic oligosaccharides formed from starch through cyclodextrin glycosyltransferase action [11]. Cyclodextrins have a hydrophilic surface and a hydrophobic core, which allows the molecules to be soluble in aqueous solutions but allows hydrophobic molecules to solubilize in cyclodextrin. Hydrophobic molecules in cyclodextrin can be solubilized in an aqueous solution, without structural change [7,10]. Pre-loaded Cholesterol (CLC) molecules have been used to incorporate cholesterol in to the sperm membranes and increase the cholesterol content of sperm membranes [20,29]. When the cholesterol content of stallion, boar, ram, and bull sperm is increased by CLC, the cryosurvival rate and quality of the frozen-thawed sperm is increased [2,15,19,20]. This decreases the membrane damage upon warming [5]; therefore, CLC
http://dx.doi.org/10.1016/j.cryobiol.2016.12.012 0011-2240/© 2016 Published by Elsevier Inc.
Please cite this article in press as: N. Chuaychu-noo, et al., Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.12.012
2
N. Chuaychu-noo et al. / Cryobiology xxx (2016) 1e5
Unless otherwise stated, all chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). The cell viability test kit was obtained from the Life Technologies (Carl.sbad, CA, USA).
(O.P.) ¼ 395 mOsm/kg) [31]. Immediately after collection of the semen sample from each breed group, these were pooled separately in 15 mL tubes. Semen in each tube was divided into 4 aliquots and diluted (to 0, 1, 2 and 3 mg/1 mL) of CLC, Schramm-based diluter 1: 3 (v: v) was then cooled to 5 C for 60 min (1 C per 3 min). At this temperature, the DMF (N,N-dimethylformamide) was added to a final concentration of 6% (v/v) and then mixed. Semen was immediately loaded into 0.5 mL plastic straws and sealed with Polyvinylpyrrolidone powder (PVP). The number of final sperm concentration in each straw was about 500 106. After 15 min of equilibration, the filled straws were laid horizontally on a rack 11 cm above the surface of liquid nitrogen (35 C) for 12 min then placed 3 cm above the liquid nitrogen vapor (135 C) for 5 min, and subsequently immersed in liquid nitrogen for storage at 196 C until analyzed. Thawing was achieved at 2-5 C for 5 min in cool water [33].
2.2. Animals
2.6. Evaluation sperm quality
Twelve Pradu Hang Dum (Thai Native) and twelve Rhode Island Red, 1 to 2-year-old breeder male roosters were housed in individual cages, fed 130 g/d/b of commercial feed, and given water ad libitum. Forty-eight individually housed, commercial laying hens (24 weeks of age) having egg production of 85e95% were fed 110 g/ d/b of layer feed and given water ad libitum. The animals were reared under natural environment conditions. The study was conducted at the research farm, Department of Animal Science, Faculty of Agriculture, Khon Kaen University; Thailand. The Animal Ethics Committee of Khon Kaen University reviewed and approved the study (Approval No.: 0514.1.12.2/29).
The concentration of sperm was determined with a haemocytometer under a light microscope. The computer-assisted sperm analysis system (CASA) (version 10 HIM-IVOS; Hamilton Thorne Biosciences, Beverly, MA USA) has the ability to assess total motility (MOT), progressive motility (PMOT), velocity average path (VAP), velocity straight line (VSL), and velocity curvilinear (VCL) [12]. The percentage viable was determined by SYBR - 14 and Propidiam Iodide (PI) (Live/Dead Sperm Viability Kit, InvitrogenTH, Thermo Fisher Scientific, Waltham, MA, USA) [24]. The percentage of acrosome integrity and functional mitochondria was determined by PI (Sigma, 81845), 5, 50 , 6, 6’ e tetraethybenzimdazolycarboncyanine iodide (JC- 1; Sigma, C50390) and fluorescein isothiocyanate conjugated peanut agglutinin (FITC-PNA; Sigma, L7381). These were evaluated by flow cytometry (FACSalibur; Becton Dickinson, San Jose, CA, USA) as described by LangeConsiglio [13] with minor modifications.
treatment prior to cryopreservation may be able to decrease rooster sperm damage after cryopreservation and increase fertility. To our knowledge, there is no published information on the effects of CLC in chicken cryopreserved semen. Our aim therefore was to determine whether treating rooster sperm with CLC prior to cryopreservation would improve post-thaw sperm survival and also to optimize the concentration of CLC suitable in the semen freezing diluent for improving fertility. 2. Materials and methods 2.1. Chemicals
2.3. Preparation of cyclodextrin Cholesterol-Loaded Cyclodextrin (CLC) was prepared as per Purdy and Graham [27]. Briefly, 1 g of cyclodextrin was dissolved in 2 mL of methanol in a glass tube. In a second glass tube, 200 mg of cholesterol was dissolved in 1 mL of chloroform. The cyclodextrin/ methanol solution was added to the cholesterol/chloroform solution and mixed (glass tube 1 into 2). The solution (in a petridish) was then subjected to a stream of nitrogen gas to vapor at the solvent and to obtain a white CLC, which was stored at 5 C. The CLC working solution was mixed with Schramm diluent, using a vortex mixer for 30 s [27]. 2.4. Semen collection Semen was collected twice a week, by the dorso-abdominal massage method [28]. Semen from an individual rooster was collected in a 1.5 mL micro-tube containing 0.1 mL of Schramm diluent. To maximize semen quality and quantity, the collection was performed by the same person, under the same conditions, at the same time, and using the massage method. Care was taken to avoid contamination of semen with feces, urates, and transparent fluid-all of which lowers the semen quality. Semen samples were selected on the basis of meeting the following criteria: mass motility score 4; sperm concentration 3 109 sperm/mL; sperm viability 85%; and, 90% normal cells in the semen sample [33]. 2.5. Semen dilution and cryopreservation Schramm diluents was used in the current study, comprising of magnesium acetate 0.7 g, sodium glutamate 28.5 g, glucose 5 g, inositol 2.5 g, and potassium acetate 5 g, all of which were dissolved in 1,000mL of double-distilled water (pH ¼ 7.1, Osmotic pressure
2.7. Lipid peroxidation Malondialdhyde (MDA) concentration as indices of the lipid peroxidation in the semen samples was measured using the Thiobarbituric acid (TBA) reaction. Semen samples from each treatment 250 mL (250 106 spz/mL) was centrifuged at 1,800 rpm for 3 min and the supernatant discarded and 1 mL of Schramm diluent added. The semen pellet was added with 0.25 mL Ferrous sulfate (Ajex, 0906251) (0.2 mM) and 0.25 mL ascorbic acid (Sigma, A5960) (1 mM). The mixture was incubated at 37 C for 60 min after which 1 mL trichloroacetic acid (Sigma, T6399) (15%) and 1 mL thiobarbituric acid (Sigma, T550) (0.375%) were added, and boiled in water for 10 min. Thereafter, the samples were cooled to room temperature to stop the reaction. Finally, the samples were centrifuged to controlled temperature of 4 C at 5,000 rpm for 10 min and analyzed, using UVeVisible spectrophotometer (Analytikjena Model Specord 250 plus) at 532 nm [24]. 2.8. Fertility The fertility ability of frozen-thawed spermatozoa was tested by inseminating layer hens (48 hens were randomly assigned to 8 groups of 6 hens) once a week with a dose of 0.4 mL. All inseminations were performed between 15.00 and 17.00 Hrs. Eggs were collected during days 2e8 after insemination. Fertility was determined by candling eggs on day 7 of incubation. Six replications of fertility test were carried out. For the fertile period test, 48 hens (about 80% of hen-day production) were randomly assigned to 3 groups, and inseminated
Please cite this article in press as: N. Chuaychu-noo, et al., Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.12.012
N. Chuaychu-noo et al. / Cryobiology xxx (2016) 1e5
with fresh semen, frozen semen (control), and the best group from experiment of CLC supplemented were tested by inseminating layer hens once a week with a dose of 0.4 mL. All insemination was performed at 15.00e17.00 Hrs. Eggs were collected during days 2e16 after insemination. Fertility was determined by candling eggs on day 7 of incubation [33]. 2.9. Statistical analysis The experiments were conducted using 2 4 factorial randomized complete block design (RCBD). Statistical analysis was performed using the General Linear Model (GLM). The mean of the CLC treatment effects were compared using Duncan's Multiple Range test (DMRT) (P < 0.05). The arcsine square root transformation was used SAS [30]. Six replications were used for evaluating sperm quality with flow cytometry, CASA, MDA, fertility test, and duration of fertility respectively. 3. Results 3.1. Effects of CLC supplementation of diluent on sperm motility and sperm viability in different breeds of rooster The effects of breeds on frozen semen quality are presented in Table 1. Rhode Island Red had a higher sperm viability than Pradu Hang Dum (P < 0.05) but there was no difference in motility between breeds. CLC supplementation (2 and 3 mg/mL) of frozen semen resulted in a higher quality over against the without control (P < 0.01). Neither breed nor CLC supplementation had any appreciable effect on percentage of MOT, PMOT, VAP, VSL, VCL. Rhode Island Red had higher live cells with intact acrosomes (P < 0.05) than Pradu Hang Dum (Table 2). Supplementation with 1, 2 and 3 mg/mL CLC in the diluents resulted in a higher percentage of live with intact acrosome (P < 0.01) than the control group. Pradu Hang Dum with functioning mitochondria was greater (P < 0.01) than Rhode Island Red and CLC supplementation (1, 2 and 3 mg/mL) was greater than the control group (Table 2). 3.2. Effects of CLC supplementation of diluent on fertility and lipid peroxidation in semen The effects of CLC supplementation of Scharmm extender on fertility in different breeds are presented in Table 2. Breeds had no effect on fertility rate; however, supplementation of 1 mg/mL CLC in the freezing extender improved the fertility of frozen semen (P < 0.01). CLC had no effect on the MDA levels in frozen semen
3
(P > 0.05). 3.3. Effects of CLC supplementation of diluent on fertile period Fertility rates were compared after Artificial Insemination (AI) using fresh semen and frozen semen, control, and 1 mg/mL CLC supplementation in diluents for 3 periods post AI (days 1e7, 8e14 and 1e14) (Fig. 1). The results showed that the fertility rate of fresh semen was the highest and that frozen semen treated with CLC was higher than the control group. Frozen semen on days 1e7 was 88.85 ± 5.23, 81.63 ± 4.40 and 73.10 ± 6.63%; on days 8e14 was 48.43 ± 1.68, 23.30 ± 4.55 and 14.14 ± 3.67%; and on days 1e14 was 66.42 ± 1.17, 54.08 ± 2.45, and 42.26 ± 3.36% respectively. 4. Discussion The current study was under taken to obtain a suitably modified semen diluent for improving the quality of the cryopreserved -vis fertility rate after AI under field consemen of chickens' vis-a ditions. Based on the results, we concluded that supplementation with 1% CLC improves post-thaw semen quality in chickens. To our knowledge, this is the first report on the effects of CLC on frozen semen quality in chickens. Rooster sperm with CLC improved sperm viability and membrane integrity of the sperm cells in a manner similar to CLC supplementation of mammalian sperms [23]. The results of the experiment show that CLC at 1, 2 and 3 mg/ mL improved rooster frozen semen quality. This is similar to the optimal CLC concentration reported for improving the quality of the frozen semen of bulls, stallions, boars, and goats [19,20,27,29,32]. Previous reports demonstrated that semen diluent supplemented with CLC improved sperm survival, motility, membrane integrity, and fertility in many farm animal species [18]. The cholesterol to phospholipid ratio is an important determinant of cryosurvival rates and is different for sperms from different species, lines within a species, males, and after various treatments [19]. These components are crucial for sperm membrane fluidity and stability at low temperatures. Nevertheless, the benefit of CLC treatments modifies the capacitation pattern, timing of sperm, and subsequent ability of sperm to undergo the acrosome reaction. Although avian spermatozoa do not seem to undergo capacitation or motility hyper-activation prior to fertilization [16], they do spend a very long time in the female oviduct before oocyte penetration [9]. Howarth [9] reported that fowl sperm have been shown to undergo acrosome reaction; thus fertilizing the ovum directly without requiring capacitation. Possible in vivo fertilization in chickens, the perivitelline layer just after ovulation is composed of
Table 1 Sperm quality parameters of frozen semen of 2 breeds of rooster at 4 concentrations of CLC supplementation (mean ± SD; n ¼ 12). Factor Breeds
VI(%)
MOT(%)
PMOT(%)
VSL(mm/s)
VCL(mm/s)
VAP(mm/s)
Pradu Rhodes
43.63 ± 4.42 45.90 ± 3.98a
57.45 ± 7.80 60.90 ± 7.52
34.24 ± 3.57 36.50 ± 3.83
76.71 ± 6.31 77.88 ± 6.04
131.90 ± 6.13 134.35 ± 6.63
91.45 ± 5.55 92.94 ± 7.89
0.03
0.21
0.08
0.53
0.23
0.50
0 1 2 3
40.60 42.62 44.67 45.04
P- value Level of CLC (mg/mL)
b
± ± ± ±
2.47C 2.91B 3.16A 4.78A
58.60 58.20 59.90 60.00
± ± ± ±
5.51 9.65 8.17 6.81
34.03 34.83 36.03 36.60
± ± ± ±
3.03 4.21 3.07 4.57
75.23 79.58 77.66 76.72
± ± ± ±
3.52 7.36 5.81 7.05
133.23 133.96 132.96 128.37
± ± ± ±
2.77 7.87 5.24 9.00
90.64 92.55 92.78 92.82
P- value
0.001
0.95
0.48
0.43
0.95
0.88
Interaction
0.57
0.68
0.39
0.59
0.50
0.62
± ± ± ±
3.36 6.75 7.27 9.75
Different letters (A,B) within columns indicate significant differences (P < 0.01). Different letters (a,b) within columns indicate significant differences (P < 0.05), VI (%): percentage of viable spermatozoa with an intact plasma membrane assessed by propidium iodide staining (PI negative); MOT (%): percentage of motile spermatozoa; PMOT (%): percentage of spermatozoa with progressive motility; VAP (mm/s): percentage of velocity average path; VSL (mm/s): percentage of velocity straight line; VCL (mm/s) percentage of velocity curvilinear.
Please cite this article in press as: N. Chuaychu-noo, et al., Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.12.012
4
N. Chuaychu-noo et al. / Cryobiology xxx (2016) 1e5
Table 2 Effects of CLC supplementation of the diluent on acrosome integrity, functional mitochondria on fertility, and malondiodehyde of rooster spermatozoa in frozen-thawed semen (mean ± SD; n ¼ 12). Live with intact acrosomes
Functional mitochondria
MDA (mm/mL/250 106spz)
% Fertility (egg set)
Pradu Rhode
27.32 ± 4.37b 29.15 ± 6.88a
38.28 ± 4.58A 36.30 ± 6.75B
1.67 ± 0.53 1.53 ± 0.33
85.73 ± 9.38 (995) 87.61 ± 7.84 (913)
0.04
0.005
0.27
0.31
0 1 2 3
20.03 29.56 30.56 31.80
Factor Breed P- value Level of CLC (mg/ml)
± ± ± ±
2.62B 1.80A 2.50A 4.54A
29.17 37.28 39.96 42.55
± ± ± ±
3.20C 3.19B 1.82AB 3.00A
1.51 1.46 1.77 1.67
± ± ± ±
0.47 0.25 0.59 0.37
85.90 91.90 84.60 82.29
P-value
0.001
0.001
0.95
0.003
AxB
0.06
0.06
0.42
0.09
± ± ± ±
7.98B (448) 4.31A (492) 9.63B (481) 9.38B (447)
Different letters (A,B) within columns indicate significant differences (P < 0.01), Different letters (a,b) within columns indicate significant differences (P < 0.05), MDA; malondiodehyde.
Fig. 1. Fertile period after one artificial insemination. Different letters (A, B) with in bar indicate significant difference (p < 0.01).
inner perivitelline membrane, which is secreted just after fertilization in the upper part of oviduct. As studied in vitro, the results demonstrated that the acrosome reaction may be induced within a minute [14]. According to in vitro experiments, in mammalian species CLC extended sperm viability and acrosome integrity both in fresh and cryopreserved forms [19]. Our result indicates that sperm motility, sperm viability and sperm viability with heightened mitochondrial function are higher in spermatozoa treated with CLC than the untreated one and this parameter was higher in those treated with 2 and 3 mg CLC than those treated with 1 mg CLC. Recently, a study of the effect of CLC on sperm viability and acrosome reaction in live semen cryopreservation showed higher cholesterol incorporation in the acrosome membrane than the controlled. CLC not only reduced the premature acrosome reaction but also protected the viability of sperm and prevented premature acrosome reaction and capacitation, usually a side effect of cryodamage [15]. The in vitro assessment of sperm characteristic is important for determining sperm fertility, but those parameters are not conclusive. Thus, a fertility trial is needed to elucidate the real effect on fertility. The reason as to why the increased sperm quality does not
translate into a higher fertilizing ability is not known [18]. This may be due to CLC-treated sperm requiring more time to capacitate in the female reproductive tract, resulting in appropriate timing of AI i.e., between the acrosome reaction of the sperm and ovulation [19]. We hypothesize that in the current study, the low fertility rate of 2 and 3 mg CLC groups may be due to acrosome reactionpreventive property of CLC, which may have interfered with the sperm's penetrating ability through the inner perivitelline membrane. Thus, supplementation of CLC (more than 1 mg/mL) may interfere with the acrosome reaction leading to a decline in fertility rates. Semen supplemented with the optimal CLC concentration thus benefitted sperm characteristics, including sperm membrane integrity, viability, and fertilizing potential. The current study was performed to determine the effects of CLC on two breeds of chicken namely, Pradu Hang Dum and Rhode Island Red. The results showed that there were differences between -vis in percentage sperm viability i.e., live with the breeds vis-a intact acrosome: the Rhode Island Red had greater viability than Pradu Hang Dum. Pradu Hang Dum had more live with high mitochondrial activity than the Rhode Island Red. There was, however, no difference between breeds on post-thawed semen
Please cite this article in press as: N. Chuaychu-noo, et al., Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.12.012
N. Chuaychu-noo et al. / Cryobiology xxx (2016) 1e5
fertility. The success of freezing poultry semen is highly variable and depends on the species and the specific breed lines [3,17,22,25,35]. The percentage of sperm viability that is live with intact acrosome and live with high mitochondrial activity is correlated to fertility [3]. The number of surviving sperm in the reproductive tract may not be different between these two breeds, but the fertilizing ability of the frozen sperm of both breeds had similar fertilization rates. Although the fertility and fertile period of fresh semen is better than the frozen semen groups, the fertility rate and fertility period of the CLC dsupplemented group was superior to the frozen semen control group (Fig. 1). The duration of fertility was correlated with the number of sperm in the SST of the UVJ [26]. The results suggest that supplementation with 1 mg/mL CLC in the freezing diluents would improve survival and longevity of sperms over against the control frozen semen. In conclusion, CLC in freezing diluent improves the quality of frozen semen and subsequent fertility rates over against nonfrozen sperm. In the current study, there was no difference among breeds of rooster on post-thaw fertility. Taken together, the results of this study suggest that 1 mg/mL of CLC in the freezing diluent is an optimal concentration for improving the subsequent fertility of chickens impregnated with the thawed frozen sperms. Statement of funding Experiment was designed by T. Vongpralub, N. Chuaychu-noo. Semen freezing and post-thawed semen evaluations were done by N. Chuaychu-noo and P. Thananurak. Artificial insemination was carried on by T. Vongpralub, N. Chuaychu-noo and P. Thananurak. Manuscript was written by T. Vongpralub and V. Chankitisakul. Acknowledgements The authors thank (1) the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Food and Functional Food Research Cluster of Khon Kaen University (URU591005) for financial support; (2) the Research and Development Network Center for Animal Breeding (Native Chicken), Department of Animal Science, Faculty of Agriculture, Khon Kaen University (3) Institute of Agricultural Technology, Suranaree University of Technology for semen quality evaluation equipments. (4) Rajamangala University of Technology Srivijaya was supported by a PhD grant. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cryobiol.2016.12.012 References [1] R.P. Amann, J.K. Graham, Spermatozoal function, in: V.O. McKinnon, J.L. Voss (Eds.), Equine Reproduction, Lea and Febiger, Philadelphia, London, 1993, pp. 715e745. [2] E.A.M. Amorim, J.K. Gramham, B. Spirizzi, M. Meyers, C.A.A. Torres, Effect of cholesterol or cholesteryl conjugates on the cryosurvival of bull sperm, Cryobiology 58 (2009) 210e214. [3] E. Blesbois, F. Seigneurin, I. Grasseau, C. Limouzin, J. Besnard, D. Gourichon, G. Coquerella, P. Rauit, M. Tixier-Boichard, Semen cryopreservation for ex-situ management of genetic diversity in chickens creation of the French Avian Cryobank, Poult. Sci. 86 (2007) 555e564. [4] E. Blesbois, U.I. Grassea, F. Seigneurin, Membrane fluidity and the ability of domestic bird spermatozoa to survive cryopreservation, Reprod 129 (2005) 371e378. [5] D.C. Bongalhardo, S. Leeson, M.M. Buhr, Dietary lipids differentially affect membranes from different areas of rooster sperm, Poult. Sci. 88 (2009) 1060e1069.
5
[6] C.A. Castellano, I. Audet, J.L. Bailey, J.P. Laforest, J.J. Matte, Dietary omega-3 fatty acids (fish oils) have limited effects on boar semen stored at 17 C or Cryopreserved, Theriogenology 74 (2010) 1482e1490. [7] H. Dobziuk, Molecules with holes e cyclodextrins, in: H. Dodziuk (Ed.), Cyclodextrins and Their Complexes, Wiley-VCH VerlagGmbH&Co, Weinheim, Germany, 2006. [8] R.H. Hammerstedt, J.K. Graham, Cryopreservation of poultry sperm: the enigma of glycerol, Cryobiology 29 (1992) 26e38. [9] J.R. Howarth, An examination for sperm capacitation in the fowl, Biol. Reprod. 3 (1971) 338e341. [10] J. Hu, Z. Tao, S. Li, B. Liu, Review: the effect of cyclodextrins on polymer preparation, J. Mater. Sci. 40 (2005) 6057e6061. [11] Z.E. Jouni, J. Zamora, M. Synder, W.R. Montfort, A. Weichsel, M.A. Wells, aCyclodextrin extracts diacylglycerol from insect high density lipoproteins, J. Lipid Res. 41 (2000) 933e939. [12] L.M. King, R.H. Denise, A.M. Donoghue, Correlation of CASA velocity and linearity parameters with sperm mobility phenotype in turkeys, J. Androl. 21 (2000) 65e71. [13] A. Lange-Consiglio, A. Meucci, F. Cremonesi, Fluorescent multiple staining and CASA system to assess boar sperm viability and membranes integrity in short and long-term exten, Open Veterinary J. 3 (1) (2013) 21e35. [14] M. Lemoine, I. Grasseau, J.P. Brillard, E. Blesbois, A reappraisal of the factors involved in in vitro initiation of the acrosome reaction in chicken spermatozoa, Reprod 136 (2008) 391e399. [15] Y.S. Lee, S. Lee, S.H. Lee, B.K. Yang, C.K. Park, Effect of cholesterol-loadedcyclodextrin on sperm viability and acrosome reaction in boar semen cryopreservation, Anim. Reprod. Sci. (2015) 124e130. [16] M. Lemoinea, S. Mignon-Grasteaue, I. Grasseaua, M. Magistrinia, E. Blesboisa, Ability of chicken spermatozoa to undergo acrosome reaction after liquid storage or Cryopreservation, Theriogenology 75 (2011) 122e130. [17] A. Massip, S.P. Leibo, E. Blesbos, Cryobiology of gametes and the breeding of domestic animals, in: E. Benson, B. Fuller, N. Lane (Eds.), Live in the Frozen State, Taylor & Francis Group, London, 2004, pp. 371e393. , E. Blanch, C. Toma s, J.K. Graham, Use of cholesterol in sperm cryo[18] E. Moce preservation: present moment and perspective to future, Reprod. Domest. Anim. 45 (2010) 57e66. [19] E. fluorescein isothiocyanate conjugated peanut agglutinin (FITC-PNA; Sigma, L7381). These were evaluated by flow cytometry P.H. Purdy, J.K. Graham, Treating ram sperm with cholesterol-loaded cyclodextrins improves cryosurvival, Anim. Reprod. Sci. 118 (2010) 236e247. [20] A.I. Moore, E.L. Squires, J.K. Graham, Adding cholesterol to the stallion sperm plasma membrane improves cryosurvival, Cryobiology 51 (2005) 241e249. [21] G.J. Morris, E. Acton, B.J. Murray, F. Fonseca, Freezing injury: the special case of the sperm cell, Cryobiology 64 (2012) 71e80. [22] T.M.B. Mosenene, Characterization and Cryopreservation of Semen of Four South African Chicken Breeds. Magister Scientiae Agriculturae, Faculty of Natural in Agricultural Sciences, University of the Free State Bloemfontein, Bloemfontein, 2009. [23] J.E. Parks, D.V. Lynch, Lipid composition and thermotropic phase behavior of boar, bull, stallion, and rooster sperm membranes, Cryobiology 29 (1992) 255e266. [24] A. Partika, W. Nizanski, E. Lukaszewicz, Evalution of fresh and frozen-thawed fowl semen by flow cytometry, Theriogenology 74 (2010) 1019e1027. [25] S.O. Peters, O.D. Shoyebo, B.M. Ilori, M.O. Ozoje, C.O.N. Ikeobi, O.A. Adebambo, Semen quality traits of seven strain of chickens raised in the humid tropics, Int. J. Poult. Sci. 7 (2008) 949e953. [26] E.E. Pierson, G.R. McDaniel, L.M. Krista, Relationship between fertility duration andin vivo sperm storage in broiler breeder hens, Brit Poult. Sci. 29 (1988) 199e203. [27] P.H. Purdy, J.K. Graham, Effect of adding cholesterol to bull sperm membranes on sperm capacitation, the acrosome reaction, and fertility, Biol. Reprod. 71 (2004) 522e527. [28] J.P. Quinn, W.H. Burrows, Artificial insemination of fowls, J. Hered. 27 (1937) 31e37. [29] B.E. Spizziri, M.H. Fox, J.E. Bruemmer, E.L. Squires, J.K. Graham, Cholesterolloaded cyclodextrins and fertility potential of stallions spermatozoa, Anim. Reprod. Sci. 118 (2010) 255e264. [30] SAS Institute Inc, SAS/STAT User's Guide: Version 6.12, fourth ed., SAS Institute Inc., Carry, North Carolina, 1966. [31] G.P. Schramm, Suitability of different antifreeze agents for cryoprotection of cock sperm, Monatsh. Fuer Veterinaermedizin 46 (1991) 438e440. z, E. Blanch, M. Hern [32] C. Toma andez, M.A. Gil, J. Roca, J.M. V azquez, , Treating boar sperm with cholesterol-loaded cycloE.A. Martínez, E. Moce dextrins widens the sperm osmotic tolerance limits and enhances the in vitro sperm fertilising ability, Anim. Reprod. Sci. 129 (2011) 209e220. [33] T. Vongpralub, A. Utha, J. Pongpeng, P. Sonseeda and W.Yindee, A comparison of simple vapour method and programmable freezer on motility and fertility of frozen Thai native chicken semen, in: The 3 rd International Conference on Sustainable Animal Agriculture for Developing Countries (SAADC 2001), July 26-29 2011, Nakhon Ratchasima, Thailand, 2011. [34] P.F. Watson, The causes of reduced fertility with cryopreserved semen, Anim. Reprod. Sci. 60e61 (2000) 481e491. [35] G.J. Wishart, Semen quality and semen storage, in: P.M. Hocking (Ed.), Biology of Breeding Poultry, CAB Int, 2009, pp. 151e178.
Please cite this article in press as: N. Chuaychu-noo, et al., Supplementing rooster sperm with Cholesterol-Loaded-Cyclodextrin improves fertility after cryopreservation, Cryobiology (2016), http://dx.doi.org/10.1016/j.cryobiol.2016.12.012