Effect of egg yolk plasma and soybean lecithin on rooster frozen-thawed sperm quality and fertility

Theriogenology 116 (2018) 89e94

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Effect of egg yolk plasma and soybean lecithin on rooster frozen-thawed sperm quality and fertility Mahdieh Mehdipour a, Hossein Daghigh Kia a, *, Gholamali Moghaddam a, Hamed Hamishehkar b a b

Department of Animal Science, College of Agriculture, University of Tabriz, Tabriz, Iran Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

a r t i c l e i n f o

a b s t r a c t

Article history: Received 6 January 2018 Received in revised form 10 May 2018 Accepted 12 May 2018

This experiment was conducted to study the effects of egg yolk plasma (10%, 15% and 20%), soybean lecithin (0.5%, 1% and 1.5%) and whole egg yolk (WEY) (control) on post-thawed sperm quality, hatchability and fertility outcomes. In experiment 1, sperm motility, abnormalities, membrane integrity, viability, apoptosis status, mitochondrial activity were studied following freeze-thawing. The best quality of frozen-thawed rooster sperm was chosen to be used for the assessment of the hatchability and fertility rate in experiment 2. The significantly higher percentages of post-thawing sperm total and progressive sperm motilities, membrane integrity, viability were observed in 1% soybean lecithin and 20% egg yolk plasma in comparison with 0.5 and 1% soybean lecithin, 10% egg yolk plasma and control, except for 15% egg yolk plasma (P < 0.05). Using 20% egg yolk plasma in the extender improved mitochondrial activity. Supplementation of 1% soybean lecithin and 20% egg yolk plasma into the extender resulted in the least percentages of dead sperm (P < 0.05). Sperm abnormalities and early apoptosis did not differ in various extender supplementations. In experiment 2, higher percentages of hatchability and fertility rate were observed in semen containing 1% soybean lecithin and 20% egg yolk plasma compared with the WEY group. The results showed that supplementation of the rooster sperm extender with 1% soybean lecithin and 20% egg yolk plasma resulted in higher quality of frozen-thawed sperm. © 2018 Elsevier Inc. All rights reserved.

Keywords: Freezing Rooster semen Artificial insemination Cryoprotectant

1. Introduction The most worthwhile technique for preservation and management of bird genetic resources is semen cryopreservation, which has been studied in domestic birds like chicken [1]. Despite years of intensive investigations, still more work should be done in order to perform successful cryopreservation of poultry sperm. The lower quality of frozen-thawed poultry sperm and consequently the poor fertilization rates compared to mammalian species are due to the exceptional morphological properties of poultry sperm, which cause freeze damages because of their vulnerable structure to low temperatures [2]. Egg yolk was quickly adopted as anon-permeable cryoprotectant that decreases the freezing point of the medium and produces less ice crystals [3]. Although it has

* Corresponding author. E-mail addresses: [email protected], [email protected] (H. Daghigh Kia). https://doi.org/10.1016/j.theriogenology.2018.05.013 0093-691X/© 2018 Elsevier Inc. All rights reserved.

been documented that the cryoprotective effect of egg yolk is attributed to low density lipoproteins, phospholipids or triglycerides [4] but the mechanism by which it protects the sperm cell during cryopreservation, is not exactly recognized. Reasonable results achieved from different studies have made egg yolk extender as a protective agent to defend sperm against the harmful effects of low temperature. Despite its benefits, several arguments have been grown against the use of egg yolk, mostly due to its proneness to contamination with animal pathogens [5]. Egg yolk consists of two major parts including plasma and granules. Plasma contains a wide amount of valuable lipids named low-density lipoproteins (LDL), whereas granules are mainly constituted of high-density lipoproteins (HDL) which are known as unfavorable structures [6]. Different investigations have shown that HDL have the ability to disturb the function of sperm [7]. It has been revealed that the part responsible for the cryoprotective properties is LDL. Some properties of egg yolk plasma such as its easy extraction and comparable effect to LDL have made it a suitable

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alternative to whole egg yolk [8]. Moreover, soybean lecithin is suggested to replace WEY for sperm cryopreservation in most of the livestock species [9,10]. It has been proposed that the lecithin protection mechanism is due to the replacement of sperm membrane phospholipids, with minimizing the freezing point. Also, it may form a protective layer around the sperm, avoiding the formation of ice crystals, consequently preventing the physical damage to the sperm membranes [11]. Extenders including soybean lecithin are successfully used worldwide for sperm cryopreservation in variety of livestock species [12e14]. However, its effects on sperm functional properties yet need to be entirely studied. Therefore, the aim of the present study was to analyze the use of egg yolk plasma or soybean lecithin instead of WEY in extenders for the cryopreservation of rooster semen. Our hypothesis was that soybean lecithin and egg yolk plasma extenders would be more effective than whole egg yolk-based extender at cryopreserving rooster sperm. This strategy might improve the protocol of rooster sperm freezing by developing a new extender before using in artificial insemination. Nevertheless, cellular mechanism of egg yolk plasma or soybean lecithin on in vivo fertility potential in rooster has not been completely clarified. Therefore, several parameters such as sperm motility, abnormalities, membrane integrity, viability, mitochondrial activity, apoptosis status, hatchability and fertility rate were assessed in this study to find the best cryoprotectant for cryopreservation of rooster sperm. 2. Materials and methods 2.1. Chemicals The chemicals used in this experiment were obtained from Sigma (St. Louis, MO, USA) and Merck (Darmstadt, Germany). 2.2. Egg yolk plasma The hen egg yolk was separated into two fractions containing plasma and granules using the technique conducted by Pillet et al. [15]. Plasma fractionation was carried out by diluting an equal volume of yolk and 0.17-M NaCl solution and stirring with a magnetic stirrer for 1 h at 4
C.Then centrifugation was carried out two times at 10,000  g for 45 min at 4
C.The supernatant containing egg yolk plasma was collected and maintained in glass containers of 2 mL at 4
C for later use. 2.3. Animal ethics, farm management and semen collection The study was carried out after approval of Animal Ethics Committee of the University of Tabriz (Tabriz, Iran). Ten Ross broiler breeders at 28 weeks of age were kept individually in cages (60 cm  60 cm  75 cm (at 20
C and 14L:10D photo period at the Poultry Unit, Animal Production Centre, University of Tabriz (Tabriz, Iran). Birds were offered fresh water and a standard commercial chicken breeder diet (2750 kcal ME/kg, 11.99% CP). They were first trained for semen collection for two consistent weeks. Then, all of the semen roosters were regularly collected twice a week. The semen was collected using the method conducted by Fattah et al. [16]. The collection was always carried out by the same person and under the same condition. The criteria in normal quality of sperm were as follows: the volume: 0.2e0.6 ml (semen volume was measured visually using a graduated collection tube); the concentration of sperm 3  109 sperm/ml (ejaculate concentration was evaluated by haemocytometer); total motility (CASA) 80% and abnormal morphology (Hancock method [17])  10%. Then, to

remove individual variations and obtain sufficient sperm for analysis, the semen samples were pooled and subsequently divided into seven aliquots in accordance with the experimental design. 2.4. Extender preparation The components of Beltsville extender contained potassium citrate tribasic monohydrate (20.8 mM), sodium-l-glutamate (512.8 mM), magnesium chloride anhydrous (35 mM), D-()-fructose (277.5 mM), potassium phosphate dibasic trihydrate (435.7 mM), potassium phosphate monobasic (51.4 mM), n-[tris (hydroxymethyl) methyl]-2 (2.71), sodium acetate tri hydrate (139.5 mM) (pH (7.2), osmolarity (310 mOsm/kg), glycerol (8%) and purified water for a total volume of 1000 mL. 2.5. Experiment 1: in vitro assessment of frozen-thawed sperm quality In experiment 1, the pooled semen samples were split into seven equal aliquots and diluted with the basic extender containing soybean lecithin (0.5%, 1% and 1.5%), egg yolk plasma (10%, 15% and 20%), and WEY (control). Then, French 0.25 mL straws were filled with the diluted semen samples with the final concentration of 400  106 sperm/mL, sealed with PVA sealing powder and equilibrated for 3 h at 5
C. Using a cryobox, the straws were arranged horizontally 4 cm above the surface of liquid nitrogen for 7 min and they were finally immersed in liquid nitrogen (196
C) for storage for further tests. For different evaluations, the straws were thawed individually at 37
C for 30 s in a water bath. 2.5.1. Motion parameters The motility parameters of sperm after thawing were evaluated using computer-assisted analyses (CASA; 12.3 CEROS, HamiltonThorne Biosciences, Beverly, MA, USA). At least 200 sperm were checked per sample using standard settings (a constant temperature of 37
C). The proportions of total motile (MOT, %), and progressive sperm (PROG, %) were specified. The motility parameters including the straight-line velocity (VSL, mm/s), the curvilinear velocity (VCL, mm/s), the average path velocity (VAP, mm/s), the linearity (LIN, %; VSL/VCL), the beat cross frequency (BCF, Hz), the straightness (STR, %; VSL/VAP), and the amplitude of lateral head displacement (ALH, mm) were determined for each sample. 2.5.2. Abnormal forms Morphology was analyzed in preparations comprising 15 mL sperm samples fixed in 300 mL Hancock solution under a phasecontrast microscope (Labomed LX400; Labomed Inc., Culver City, CA, USA) at magnification of 1000. A total of 200 sperm in each sample were checked for the percentage of total sperm abnormalities (head abnormalities, detached heads, abnormal midpieces and tail defects). 2.5.3. Membrane integrity Hypo-osmotic swelling (HOS) assay, a previously conducted method [13], was used to estimate the alterations in functional integrity based on swollen and curled tails. The test was performed by incubating a part of semen (10 mL; including 400  104 sperm) with 100 mL of hypo osmotic solution (100 mOsm/kg; sodium citrate 1.9 mM, fructose 5.0 mM) at 37
C for 30 min. Afterward, a phase-contrast microscopy (Labomed LX400; Labomed Inc., Culver City, CA, USA) was used to count 200 sperm per slide and the percentage of sperm with swollen and curved tails was determined.

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2.5.4. Sperm viability Sperm viability was estimated by eosinenigrosine stain method conducted by Fattah et al. [16]. The stain procedure was followed by mixing 10 mL of semen with 20 mL of stain on a warmed slide and immediately shedding the stain with another slide. After drying, the viable and non-viable sperm were checked by phase-contrast microscopy at magnification of 400. Sperm that showed purple staining were classified as non-viable and cells that showed a rigorous exclusion of coloration were considered viable. 2.5.5. Flow cytometric analyses Cell analyses were performed with a FACS Calibur FC (Becton Dickinson System, San Jose, CA, USA) flow cytometer equipped with standard optics, an argon-ion laser performing at 488 nm. Green fluorescence (Rhodamine-123 and Annexin-V) was set with the FL1 band-pass filter (530/30 nm) and red fluorescence (propidium iodide) was measured using the FL-3 long-pass filter (610 nm). Acquisitions were carried out using the CellQuest 3.3 software (Becton Dickinson). A total of 10000 events were acquired for each sample. 2.5.5.1. Mitochondrial activity. Rhodamine 123 and PI were applied to assess the mitochondrial activity of sperm. Five mL of R123 solution (0.01 mg/ml stock) and PI solution (1 mg/ml stock) were added to 250-mL diluted semen samples (the concentration of 50  106 sperm/ml), and then they were incubated at 37
C for 15 min in the dark before flow cytometry. The mitochondrial activity was determined by measuring the percentage of sperm with R123 high fluorescence and no PI fluorescence. 2.5.5.2. Apoptosis status. For evaluation of sperm apoptosis [13], sperm was washed in calcium buffer and readjusted to 100 mL at 1  106 sperm/mL, then 10 mL Annexin VeFITC (A; 0.01 mg/mL stock) was added. After incubating for at least 20 min at room temperature, 10 mL of propidium iodide (PI; 1 mg/mL stock) was added, again they were incubated for 10 min before flow cytometry. Sperm were organized into four different groups including viable non-apoptotic (A-/PI-); early apoptotic (Aþ/PI-); late apoptotic (Aþ/ PIþ); and necrotic (A-/PIþ) cells. The last group contained late apoptotic and necrotic cells which were categorized together as dead cells. 2.6. Experiment 2: in vivo fertility and hatchability evaluation Artificial insemination was executed using the technique conducted by Borghei-Rad et al. [18], with minor modifications. For this experiment, 30Ross broiler breeder hens were classified into three groups (n ¼ 10 hens/each group), allocating individual cages (70  70  85 cm). For conducting artificial insemination, three experimental groups were chosen using the results observed in invitro sperm trial. Soybean lecithin 1%, egg yolk plasma 20 %and control group (WEY) were applied to define the potential fertility of rooster freeze-thawed sperm. For 2 weeks, 0.25 ml of semen (100  106 sperm) were used for insemination, it was done twice a week. For each group, a total of 50 eggs in 2 weeks were selected for incubation. The eggs were placed in the setter until 18 d of incubation, afterward they were transferred to hatchery for 3 days. Fertility rate (% fertile/incubated eggs) was measured by candling the eggs at 7th days of incubation. Hatchability of fertile eggs was calculated after 21days (% hatched eggs/fertilized eggs). 2.7. Statistical analysis Data were analyzed using Statistical Analysis System software (SAS®, version 9.2). Data are presented as means ± SEM (standard

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error of the mean). Differences were considered to be significant when their probability of occurring by chance was less than 5% (p < 0.05). Treatment groups were compared for differences using Tukey test. The acquired data were analyzed using MIXED model analysis of variance (ANOVA). All analyses of hatchability and fertility data were performed using GENMOD procedure. 3. Results The effects of soybean lecithin and egg yolk plasma-based on sperm motility and kinematic parameters of frozen-thawed rooster sperm are presented in Table 1. In general, the freezing extender supplemented with 1% soybean lecithin and 20% plasma egg yolk plasma resulted in higher percentages of total and also progressive motilities, in comparison with the groups including 0.5 and1% soybean lecithin, 10% egg yolk plasma and control (P < 0.05) following the freeze-thawing process. Soybean lecithin and egg yolk plasma supplementation did not provide any significant effect on the post-thaw rooster sperm kinematic parameters (VSL, VCL, VAP, ALH, STR and BCF and LIN), in comparison with the control group. Different treatments did not affect abnormal forms after thawing. The analysis demonstrated that the samples supplemented with 1% soybean lecithin and 20% egg yolk plasma led to higher percentages of plasma membrane integrity and viability in comparison with 0.5 and 1% soybean lecithin, 10% egg yolk plasma and control, except for 15% egg yolk plasma (Table 1). Furthermore, 0.5% soybean lecithin produced the lowest significant (P < 0.05) percentage of membrane integrity in comparison with other groups. The results proved that 20% egg yolk plasma led to higher percentage of mitochondrial activity in comparison with 0.5 and 1% soybean lecithin, 10 and 15% egg yolk plasma and control, except for 1.5% soybean lecithin (Table 1). As presented in Table 1 and 1% soybean lecithin and 20% egg yolk plasma resulted in higher percentage of live post-thawed sperm compared with 0.5 and 1% soybean lecithin, 10% egg yolk plasma and control, except for 15% egg yolk plasma (P < 0.05). Supplementation of 1% soybean lecithin and 20% egg yolk plasma to extender showed the lowest percentage of dead sperm. No significant effect was noted on the percentages of early apoptosis sperm for Soybean lecithin and egg yolk plasma. The data related to the fertility and hatching rate following insemination with frozen-thawed sperm are shown in Table 2. The significantly higher rates of fertility and hatching were observed in 1% soybean lecithin and 20% egg yolk plasma compared to control group. 4. Discussion The replacement of egg yolk in diluents for freezing rooter semen is a major priority in sperm cryopreservation. The present study increases our knowledge on the effects of using egg yolk plasma and soybean lecithin in semen freezing on sperm functional properties and hatchability and fertility rate. Our results obviously show that the cryopreservation of rooster semen in the Beltsvillebased extender supplemented with 20% egg yolk plasma extender and 1% lecithin provides a superior sperm protection compared with control group. For many years, hen egg yolk has regularly been used in extenders to cryopreserve the semen of several livestock animals; however, it has involved numerous drawbacks [19]. Using egg yolk plasma was demonstrated to be an efficient replacement for WEY in frozen semen extender [15]. Earlier studies have shown some unfavorable properties for whole egg yolk such as sanitary and practical disadvantages, inconsistent composition, cryoprotectant

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Table 1 Effects of different extenders on motile parameters, viability, plasma membrane integrity, abnormal forms, mitochondria activity, live, early apoptosis and dead (mean ± SEM) of rooster sperm (n ¼ 50; 5 ejaculates/10 roosters). Parameters

Treatments

MOT (%) PROG (%) VAP (mm/s) VSL (mm/s) VCL (mm/s) LIN (%) STR (%) ALH (mm) BCF (Hz) Viability (%) Plasma membrane integrity (%) Abnormal forms (%) Mitochondria activity (%) Live (%) Early apoptosis (%) Dead (%)

SEM

control

Soybean lecithin (%)

Egg yolk plasma (%)

0.5

1

1.5

10

15

20

50.09bc 18.43cd 31.98 16.4 58.97 27.93 51.58 4.21 14.82 54.20bc 49.69b 17.27 43.31d 41.10c 22.11 36.78a

41.79c 15.73d 31.73 16.41 56.7 29.21 52.76 4.3 14.34 43.04c 39.11c 19.55 41.39d 39.75c 24.16 36.08a

68.74a 31.18a 37.28 19.35 60.95 32.07 53.03 4.16 16.48 73.56a 64.68a 13.93 61.65ab 59.32a 17.8 22.87b

50.61bc 19.14cd 33.65 17.2 58.34 29.91 51.04 4.43 15.13 55.70bc 47.28bc 17.77 46.85cd 45.95bc 25.7 28.33ab

53.76b 23.96bc 34.69 17.72 59.39 29.86 51.38 4.05 15.37 56.76bc 50.57b 16.68 47.71cd 42.73bc 21.38 35.87a

59.50ab 28.03ab 35.03 18.55 61.94 30.01 52.82 3.94 15.84 64.96ab 54.99b 15.89 55.49bc 54.77ab 15.61 29.61ab

70.12a 32.87a 37.85 20.51 62.87 32.67 54.21 3.79 16.47 76.03a 64.69a 13.47 66.26a 62.85a 16.24 20.90b

2.38 1.39 1.44 1.32 1.86 2.5 3.72 0.3 1.43 3.15 2.04 1.65 2.31 2.71 3.41 2.38

Total motile (MOT, %), and progressive spermatozoa (PROG, %) were determined. The motility parameters including the straight-line velocity (VSL, mm/s), the curvilinear velocity (VCL, mm/s), the average path velocity (VAP, mm/s), the linearity (LIN, %; VSL/VCL), the beat cross frequency (BCF, Hz), the straightness (STR, %; VSL/VAP), and the amplitude of lateral head displacement (ALH, mm) were determined for each sample. Viability (%, nigrosin-eosin method), Plasma membrane integrity (%, HOS-test), abnormal forms (%, Hancock solution), mitochondrial activity, live (%, AnnexinV-/PI-), early apoptosis (%, AnnexinVþ/PI-) and dead (%, PIþ) parameters were analyzed. Freezing medium contained Beltsville extender supplemented with whole egg yolk (control), soybean lecithin (0.5%, 1% and 1.5%), and egg yolk plasma (10%, 15% and 20%). Different superscripts within the same row indicate significant differences among groups (p < 0.05).

antagonists, HDL, and egg yolk granules that interfere with sperm motility [20]. Almost all of the disadvantages presented can be decreased with the use of egg yolk plasma fraction [15]. It has been proved that LDL (specifically, their phospholipids) are greatly recognized to be the cryoprotective factor in egg yolk. Therefore, LDL have been extracted from plasma by different methods and the quality of their frozen semen in different species was investigated, obtaining promising results [21]. Also, Shahverdi et al. [22] reported that supplementation of the Beltsville extender with 4% LDL resulted in higher quality of frozen-thawed rooster sperm. It is obvious that the protocol of LDL extraction has some technological and sanitary limitations since it is laborious and time consuming, and could never be reproduced on an industrial scale nor sterilized in accordance with bio-health standards and LDL still contain products of animal origin. In contrast to LDL, egg yolk plasma is faster, easier and cheaper to be extracted from the whole egg yolk. However, LDL cannot be ordinarily used in freezing extenders, as they are not industrially produced and sterilized [15]. Therefore, we studied egg yolk plasma instead of LDL which has the problems mentioned above. In the present study, 20% egg yolk plasma extender and 1% lecithin supplementation into freezing extender improved postthawed sperm motility. Hussain Shah et al. [23], reported that adding 20% egg yolk plasma to water buffalo sperm freezing extender led to a higher percentage of sperm motility, mitochondrial activity and membrane integrity after thawing.

Table 2 Effect of different extenders on fertility and hatchability rates of rooster semen after freeze-thawing (n ¼ 50; 5 ejaculates/10 roosters). Treatments

Fertility (%)

Hatchability of fertile egg (%)

control soybean lecithin 1% egg yolk plasma 20%

38b 54a 60a

48.11b 74.07a 83.33a

Different superscripts letters within column are significantly different (P < 0.05). Freezing medium contained Beltsville extender supplemented with whole egg yolk (control), soybean lecithin 1%, and egg yolk plasma 20%.

It was reported that dog sperm extended with 20% egg yolk plasma could be kept cool for up to 10 h prior to freezing and resulted in significantly higher sperm motility, membrane and acrosome integrity post-thawing, so it could be used as an efficient cryoprotective media [24]. Also, Kampschmidt et al. [25], reported that the granules found in egg yolk can decrease bull sperm respiration and subsequently motility. Therefore, in the present study, the improved motility might be due to the granules removal from egg yolk and using egg yolk plasma instead. It was proved that the incorporation of LDL in bovine extenders led to the same path velocity, progressive velocity and curvilinear velocity in comparison with extender containing 20% egg yolk [4]. In agreement to our results, Forouzanfar et al. [26], reported that sperm freezing in soybean lecithin containing a tris-based extender including 1% lecithin resulted in higher sperm motility and viability and cleavage rates. Papa et al. [27] reported that the soybean lecithin extender preserved stallion sperm motility similar to the conventional extender containing egg yolk. It was proved in a study that the motility of equilibrated ram semen in the soybean lecithin was significantly lower than that of observed in the egg yolk extender groups [28]. It is believed that lecithin has neither cytotoxic effect nor negative effect on sperm motility [29]; however, in our trial, high and low doses had negative effects on sperm motility after freezing and thawing but the appropriate level resulted in improving the quality of preserved rooster sperm. Similar results have been reported by other investigators, who stated that adding appropriate level of soy lecithin declines osmotic pressure owing to the precipitation of fructose and salts included in the extender, and this reduction of osmotic stress decreases the damage of sperm cells [11]. In the present study, supplementation of extender with 1.5% soybean lecithin could not improve the quality of post-thawing sperm parameters like motility and viability. This event may be due to the high viscosity of 1.5% soybean lecithin supplementing in the extender. It is mentioned that high concentrations of lecithin can enhance the viscosity of extenders and it is also noted that particular debris in the extender may have a negative effect on

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post-thawing sperm motility [30]. In this study, the percentage of total abnormality in post-thawed sperm was not affected by cryoprotectants. Cold shock during semen cryopreservation may bring about some abnormalities in sperm [31]. It has been reported that there was no significant difference in sperm morphology after freeze-thawing process in extenders containing soy soybean lecithin [32]. It was also observed in the present study that sperm cryopreserved in 20% egg yolk plasma showed higher percentages of live sperm compared with those cryopreserved in the extender containing whole egg yolk (control). Although the underlying mechanism responsible for protecting sperm by soybean lecithin during freezing/thawing is poorly understood. Two hypotheses have been proposed to explain it. Soybean lecithin can enter the cell to decrease the freezing point of ice crystals by replacing plasmalogens and relieve mechanical damage to bio-membranes of sperm [33]. Also, it is believed that soybean lecithin phospholipids integrate with sperm membrane to reduce the possible mechanical damage to the sperm membrane during the freeze-thawing and prevent the formation of lethal intracellular ice crystals by forming a protective layer around the sperm cell [34]. Therefore it is reasonable that in the present study, 1% soybean lecithin and 20% plasma resulted in a higher percentage of sperm with functional membrane integrity. It is also proposed that LDL existing in egg yolk plasma could incorporate into the plasma membrane and replace sperm membrane phospholipids that are lost or damaged during freezing [35]. Moreover, it is suggested that WEY contains substances that decrease the quality and fertilizing capacity of sperm, which could reduce fertility [5]. The results showed that 20% egg yolk plasma and 1% lecithin contained in the extender led to higher percentage of viability compared to control. The higher acrosin activity in the egg yolkebased extender would suggest membrane damage, as confirmed by the lower rates of viable sperm [36]. Another study suggested that it could be related to the high-density lipoproteins in egg yolk, which cause a decline in the semen quality by producing an efflux of cholesterol from the sperm plasma membrane and lead to alteration in fluidity that enhances the sensitivity of the membrane to cold shock [37]. Also, our finding is inconsistent with the result of Ustuner et al. [28], who reported that whole egg yolk was superior to lecithin in supporting post-thaw sperm viability. In the present study, supplementation of 20% egg yolk plasma to extender led to the protection of mitochondrial function as it prevented the fast rate of decline for mitochondria activity during cryopreservation. Energy is stored in the mitochondria as a proton concentration gradient and an electric potential gradient across the membrane [38]. Alterations in mitochondria are regarded as diagnostic predictors of sperm motility. Sperm cells are mainly based on extracellular substrates such as glycerol, lactic acid, amino acids, mono saccharides, and fatty acids to supply their energy requirements. Endogenous substrates contribute only approximately 10% of the generated energy and ATP [39]. Almost 60% of the energy available from ATP is consumed to maintain motility [40]. Annexin-V/PI test was performed to reliably estimate phosphatidyl serine (PS) translocation as an indicator of apoptotic-like changes developed during cryofreezing. This index has been known to increase during cryopreservation [41]. In agreement with our results, Emamverdi et al. [11] indicated that lecithin extenders resulted in significantly lower percentage of early apoptotic sperm than that in egg yolk extender. In the present trial, supplementation of 20% egg yolk plasma and 1% lecithin in extender significantly increased viable and decreased dead post-thawed rooster sperm compared to other extenders. Therefore, some parts of the positive effects of 20% plasma egg yolk extender and 1% lecithin on total and progressive motility may be due to higher viable and lower dead

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sperm after thawing. The fertility and hatching rates obtained with cryopreserved sperm in extender containing 20% egg yolk plasma extender and 1% lecithin were 54% and 60%, respectively. Such rates are comparable to fertility studies using cryopreserved sperm for artificial insemination [42,43]. There was a significant improvement in fertility rate with 20% egg yolk plasma extender and 1% lecithin compared to control group. This improvement is due to the higher quality of sperm treated with 20% egg yolk plasma extender and 1% lecithin, which can directly influence the results of fertility. Our fertility results with addition of 20% egg yolk plasma extender and 1% lecithin to extender were similar to the results reported in an earlier fertility trial experiment [18,43]. Masoudi et al. [44], indicated that soybean lecithin can successfully be used instead of egg yolk as a supplement for ram cryopreservation medium, without any adverse effects on post-thawed sperm quality and fertility. Moreover, Akhter et al. [45]reported that adding soy-lecithin in extender improves the freezability and fertility of buffalo bull sperm and can be used as an alternative to egg yolk in cryopreservation of buffalo sperm. Some researchers suggested that reduced fertility is due to the higher viscosity and the presence of particles in extenders [33]. Furthermore, Vishwanath and Shannon [46], reported that the particulate matter in egg yolk globules impedes the microscopic analysis of semen. Our study proves these results; moreover, physical properties of the extender might affect sperm characteristics and consequently can influence the success of artificial insemination. 5. Conclusion It is the first study that investigated a proper cryoprotectant (egg yolk plasma or soybean lecithin) for replacing egg yolk in rooster cryopreservation medium. We concluded that the extender containing 20% egg yolk plasma extender and 1% lecithin can be good alternatives to conventional extenders that contain WEY. Also, 20% egg yolk plasma extender and 1% lecithin improved fertility and hatchability rates. Despite the precise composition and validation of post-thaw semen quality and fertility, the cost and nonavailability of lecithin have limited the extensive use of soy-based extender as the method of choice in the animal artificial insemination industry. Therefore, we suggested that whole egg yolk in a cryopreservation extender could be replaced by 20% egg yolk plasma extender, in industrially or ordinary scalewithout detrimental effects on post-thawing rooster sperm quality. Conflicts of interest None of the authors have any conflict of interest to declare. References [1] Blesbois E. Current status in avian semen cryopreservation. World’s Poult Sci J 2007;63:213e22. [2] Long J. Avian semen cryopreservation: what are the biological challenges. Poultry Sci 2006;85:232e6. [3] Farstad W. Assisted reproductive technology in canid species. Theriogenology 2000;53:175e86. [4] Moussa M, Martinet V, Trimeche A, Tainturier D, Anton M. Low density lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozenethawed bull semen. Theriogenology 2002;57:1695e706. [5] Bousseau S, Brillard J, Marquant-Le Guienne B, Guerin B, Camus A, Lechat M. Comparison of bacteriological qualities of various egg yolk sources and the in vitro and in vivo fertilizing potential of bovine semen frozen in egg yolk or lecithin based diluents. Theriogenology 1998;50:699e706. [6] Anton M. Egg yolk: structures, functionalities and processes. J Sci Food Agric 2013;93:2871e80.
ska A, Kinder M, Fraser L, Strzezek _ [7] Dziekon J, Kordan W. Metabolic activity of boar semen stored in different extenders supplemented with ostrich egg yolk lipoproteins. J Vet Res 2017;61:127e33.

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