The replacement of fresh egg yolk by lyophilized egg yolk in Tris-base extender in cryopreserved Boer and Saanen semen
Abstract
Egg yolk is a common cryoprotectant that can be used as a semen extender to protect the spermatozoa from damage during cryopreservation. Therefore, the aim of this study was to investigate the efficacy of fresh and lyophilized egg yolk, as a Tris-base extender, on the quality of cryopreserved goat semen. Semen from 10 rams of two different breeds (Boer and Saanen) was collected using an artificial vagina. Each ejaculate sample was divided into four equal aliquots, which contained 20% of the fresh egg yolk (a control group), and then 10%, 15%, and 20% of the lyophilized egg yolk as a Tris-base extender. Sperm motility and kinetic parameters were determined using a computer-assisted semen analyser. The results showed that the addition of 20% of the fresh egg yolk in Tris-base extender exhibited significantly higher progressive motility, progressive fast motility, distance curve line, and beat-cross frequency parameters in the post-thaw Boer and Saanen goat sperm when compared with the addition of 10%, 15%, and 20% of the lyophilized egg yolk. The percentage of total motility and immotile parameters in the post-thaw Boer and Saanen goat sperm were not significantly different between the control and 10%, 15% as well as 20% of the lyophilized egg yolk groups. Moreover, the percentage of viability parameter in the Boer and Saanen goat sperm was not significantly different between the control and 10% of the lyophilized egg yolk group but showed significant difference between the control group and 15% and 20% of the lyophilized egg yolk groups. Furthermore, the interaction between the two breeds was significantly different in terms of head activity and straightness parameter. In conclusion, the treatment with 20% of fresh egg yolk in Tris-base extender is superior to the lyophilized egg yolk. However, an addition of 10% of the lyophilized egg yolk in Tris-base extender presented the percentage of total motility and viability parameters showing no difference with 20% of fresh egg yolk. Therefore, 10% of the lyophilized egg yolk in Tris-base extender provided detail of the lyophilized egg yolk protocol in cryopreserved goat semen as an example of an alternative extender to 20% of fresh egg yolk for situations where an animal's origin represents a microbiological risk.
1 INTRODUCTION
The cryopreservation of sperm is an assisted reproduction technique for the establishment of a genetic resource bank and accelerates the spread of genetically superior animals by artificial insemination (AI). However, inappropriate cryopreservation causes negative effects on the physiology of sperm showing death or low fertility (Bailey et al., 2000; Jl et al., 2003). The accomplishment of semen cryopreservation depends on various factors that affect sperm quality such as breed species, age, season, sperm collection method, collection season, extender composition, cooling rate, equilibration time, freezing rate, and thawing rate (Al-Ghalban et al., 2004; Arrebola & Abecia, 2017; Gangawar et al., 2016; Purdy, 2006; Sharma & Sood, 2020). Although the use of extenders for sperm cryopreservation has dramatically improved the sperm quality, the conditions in ruminants are still suboptimal. (Lodhi et al., 2008). So far, various extenders have been developed to reduce cryodamage and to enhance post-thaw viability. Egg yolk is a common active cryoprotectant, which acts as a reservoir of phospholipids and cholesterol that are used as a conventional semen extender to protect the plasma membrane and acrosome, to prevent the harmful effects of cooling and freezing, and to enhance sperm fertilization ability in domestic livestock (Bergeron & Manjunath, 2006; Forouzanfar et al., 2010; Hu et al., 2010; Nishijima et al., 2015; Swelum et al., 2018). Extenders, based on 20% egg yolk, are usually used to cryopreserve the sperm of cattles, buffaloes and pigs (Bathgate et al., 2006; Oldenhof et al., 2010). Unfortunately, previous research reported that goats are a unique species among domestic livestock because their seminal plasma contains a special egg yolk–coagulating enzyme (phospholipase A2 and glycoprotein lipase), which derives from the secretions of the bulbourethral glands. This enzyeme can interact with egg yolk and impair spermatozoa viability (Lv et al., 2018; Purdy, 2006). Therefore, the seminal plasma is removed from the sperm by centrifugation when extenders based on egg yolk are used (Leboeuf et al., 2000; Nuti, 2007; Sharma & Sood, 2020). Nevertheless, some researchers concern that the removal of seminal plasma may cause sperm damage and is also a time-consuming process. Fortunately, alternative extenders, which reduce the sperm and egg yolk-coagulating enzyme interactions, have been developed (Azerêdo et al., 2001). Recently, a group of researchers reported that extenders supplemented with 20% egg yolk and 2% soybean lecithin in a Tris-based extender show similar efficacy on spermatozoa preservation as the method without the removal of the seminal plasma (Sun et al., 2020). However, fresh egg yolk has a limitation to apply into the extender, but using the lyophilization (freeze-drying process) involving freezing and dehydration to maintain stability for prolonged intervals is well known and a widely used for conservation of biological products such as egg yolk. Lyophilized egg yolk has been applied in ram semen as an extender to mitigate the limitation of using fresh egg yolk preparation in an extender, and exhibits similar efficacy as those of fresh egg yolk extender (Alcay et al., 2015). In addition, lyophilized egg yolk is an alternative cryoprotectant when egg yolk is contaminated by poultry diseases. A freeze-dried egg yolk based extender has been used as an extender base for applying various antioxidants to improve the quality of post-thaw goat semen (Alcay et al., 2016). However, to date no literature reveals the the performance of lyophilized egg yolk in different concentrations and the optimal level of lyophilized egg yolk based extenders for goat semen cryopreservation. Therefore, the aim of this study is to investigate the efficiency between fresh and lyophilized egg yolk in Tris-base extender on the quality of cryopreserved goat semen in different breeds.
2 MATERIALS AND METHODS
2.1 Ethics approval and consent to participate
The care and use of animals were regulated by the Center of Animal Use and Care Committee of University of Phayao, Thailand, and their rules were strictly complied with (approval No. UP-AE 63–01-04-0011).
2.2 Animal management and semen collection
Ten rams from two different breeds (Boer and Saanen) at an age of 2–4 years were used in this study. Semen was weekly collected with two ejaculations of each ram using an artificial vagina for three successive weeks. After collection, the semen was transferred to a laboratory and kept at 35°C in a water bath. The ejaculate of the individual rams was evaluated for volume, concentration and percentage of motility. The following minimum standards of fresh semen were used: at least 75% motility, at a volume of 0.7–2.0 ml, and 2.5 × 109 sperm/ml.
2.3 Preparation of semen extender and processing
The elements of extender media applied for goat semen cryopreservation contained up to 20% of fresh egg yolk and 10%, 15%, 20% of lyophilized egg yolk in Tris-base extender (150 mM tris-(hydroxymethyl)-aminomethane, 47.5 mM citric acid, 14 mM glucose, 6% (v/v) glycerol, benzyl penicillin (2000 IU/ml), and streptomycin sulphate (1 mg/ml)). Each specimen was split into four aliquots and diluted with extenders containing 20% of fresh egg yolk (control group) and 10%, 15%, 20% of the lyophilized egg yolk. The lyophilized egg yolk was crushed using a mortar and filtered with a stainless steel sieve to separate the fine lyophilized egg yolk powder. Fresh and lyophilized egg yolk were added and dissolved into the Tris-base extender. The lyophilized egg yolk in the extender was mixed by using magnetic stirrer and passed through with the use of a 25 G (0.5 × 25 mm) sterile needle with 10-ml syringe as well as a centrifuge at 1,500 g for 5 min at room temperature for sediment (granules) elimination. The supernatant (plasma) was collected. Furthermore, the homogenized extenders of fresh and lyophilized egg yolk were measured kinematic viscosity at the temperature 40°C using a Cannon-fenske routine viscometer and determined density at the temperature 25°C using the calibrated volumetric glassware and a 4-digits balance. Then after, diluted semen aliquots were packed into 0.25 ml straws, cooled to 4°C within 1.5 hr and equilibrated for 1.5 hr at 4°C. The semen straws were placed 5 cm above liquid nitrogen (LN2) for 15 min and then immediately dipped in LN2 for storage at −196°C.
2.4 Evaluation of sperm motility parameters
Semen straws (n = 3 per freezing group) were thawed at 37°C for 30 s in a water bath. After thawing the frozen semen straws, the aliquots of 3 μl of thawed semen in the 20% fresh egg yolk group (control group) and the different concentrations of lyophilized egg yolk (10%, 15%, and 20%) were pipetted and loaded into pre-heated slides (Leja Slides, SCA®, Spain) at 37°C. The semen motility and kinetic parameters were measured using CASA (Androvision® CASA Software Version 1.0.0.9, Minitube, Germany). The total motility, progressive motility, progressive fast motility, curve-line velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), distance curve line (DCL), distance straight line (DSL), distance average path (DAP), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), head activity (HAC), wobble (WOB), linearity (LIN), and straightness (STR) were measured.
2.5 Measurement of sperm viability and membrane integrity
Sperm viability was observed by eosin-nigrosin staining. The stain procedure was performed by mixing semen with 1% eosin for 30 s, adding 5% nigrosin for 30 s on a pre-warmed slide, and immediately shedding with another slide. The live and dead sperm were observed using phase-contrast microscopy. Sperm with an unstained head were considered as the live sperm and sperm having pink head were regarded as dead sperm (Inyawilert et al., 2021). The membrane integrity of spermatozoa was observed with hypo-osmotic swelling test by adding 50 μl of semen into 500 μl of hypo-osmotic solution (150 mOsm/kg) and incubating for 45 min at 37°C. A total of 200 spermatozoa were examined under a light microscope. The number of spermatozoa was classified with presence or absence of coiled tail (Longobardi et al., 2020).
2.6 Statistical analysis
The data were analysed statistically with the General Linear Model procedure using analysis of variance (ANOVA) in R software. The results were presented as mean ± SEM, and p-values < .05 were considered statistically significant with Duncan's multiple-range test.
3 RESULTS
3.1 The influence of breeds (Boer and Saanen) on the sperm quality parameters of cryopreserved goat semen
To investigate the quality of post-thaw Boer and Saanen sperm treated with fresh egg yolk and different concentrations of lyophilized egg yolk, we used CASA to analyse their kinetic parameters. The results showed that the percentage of total motility was decreased (p < .05) in Saanen. In contrast, the percentage of progressive motility, immotile in Saanen were significantly (p < .05) higher than those in Boer while the percentage of viability, membrane integrity, progressive fast motility, VCL, VSL, VAP, DCL, DSL, DAP, ALH, BCF, HAC, WOB, LIN, and STR were not different between the breeds (Table 1).
| Parameters | Breeds | |
|---|---|---|
| Boer | Saanen | |
| Viability (%) | 59.33 ± 2.51 | 57.00 ± 2.45 |
| Membrane integrity (%) | 34.50 ± 3.08 | 32.33 ± 2.37 |
| Total motility (%) | 57.43 ± 4.48a | 52.37 ± 3.18b |
| Progressive motility (%) | 29.89 ± 5.57b | 38.83 ± 4.71a |
| Progressive fast motility (%) | 11.62 ± 3.20 | 13.77 ± 2.72 |
| Immotile (%) | 42.57 ± 4.48b | 48.65 ± 3.18a |
| VCL | 49.88 ± 7.35 | 55.18 ± 6.54 |
| VSL | 21.76 ± 2.69 | 23.86 ± 2.47 |
| VAP | 27.13 ± 3.37 | 30.00 ± 2.96 |
| DCL | 11.53 ± 1.56 | 12.62 ± 1.22 |
| DSL | 3.88 ± 0.56 | 4.33 ± 0.48 |
| DAP | 5.42 ± 0.72 | 6.08 ± 0.57 |
| ALH | 0.56 ± 0.07 | 0.62 ± 0.06 |
| BCF | 5.41 ± 0.89 | 6.06 ± 0.63 |
| HAC | 0.19 ± 0.02 | 0.21 ± 0.03 |
| WOB | 0.56 ± 0.02 | 0.56 ± 0.02 |
| LIN | 0.46 ± 0.02 | 0.45 ± 0.02 |
| STR | 0.80 ± 0.01 | 0.79 ± 0.01 |
- Abbreviations: ALH, amplitude of lateral head displacement; BCF, beat-cross frequency; DAP, distance average path; DCL, distance curve line; DSL, distance straight line; HAC, head activity; LIN, linearity (VSL/VCL); STR, straightness (VSL/VAP); VAP, average path velocity; VCL, curvilinear velocity; VSL, straight-line velocity; WOB, Wobble (VAP/VCL).
- a,bValues in the row with different letters indicate significant differences (Duncan's multiple-range test).
3.2 The fresh and different levels of lyophilized egg yolk in Tris-base extender effect on quality of cryopreserved goat semen
The effect of the fresh and different levels of lyophilized egg yolk in Tris-base extender on the sperm quality parameters of cryopreserved goat semen was investigated regardless of the breeds. The results showed that the viability, total motility, and HAC of spermatozoa in the control group and in the 10% of the lyophilized egg yolk group was not significantly different. However, these parameters significantly decreased in the 15% and 20% of the lyophilized egg yolk groups (p < .05) (Table 2). The membrane integrity and immotile were not significantly different in the control group when compared with 10% and 15% of the lyophilized egg yolk groups. Furthermore, the percentage of progressive motility, progressive fast motility, VCL, VSL, VAP, DCL, DSL, DAP, ALH, and BCF in the control group was significantly higher (p < .05) than those in the 10%, 15%, and 20% of the lyophilized egg yolk groups. While WOB and LIN parameter in the control group were lower (p < .05) than those in the 10%, 15%, and 20% of the lyophilized egg yolk groups (Table 2).
| Parameters | Treatments | |||
|---|---|---|---|---|
| Control | 10% of lyophilized | 15% of lyophilized | 20% of lyophilized | |
| Viability (%) | 65.00 ± 1.85a | 62.00 ± 1.59ab | 54.17 ± 2.12bc | 51.50 ± 2.23c |
| Membrane integrity (%) | 40.17 ± 2.65a | 35.83 ± 2.23a | 33.00 ± 2.11ab | 24.67 ± 2.09b |
| Total motility (%) | 60.67 ± 5.10a | 55.21 ± 3.97ab | 53.11 ± 3.68b | 53.23 ± 3.64b |
| Progressive motility (%) | 46.15 ± 3.45a | 31.92 ± 2.61b | 26.63 ± 2.14b | 24.91 ± 2.11b |
| Progressive fast motility (%) | 20.79 ± 6.55a | 11.10 ± 4.95b | 9.11 ± 4.07b | 7.77 ± 4.00b |
| Immotile (%) | 39.33 ± 5.10b | 44.79 ± 3.97ab | 46.88 ± 3.68a | 46.79 ± 3.64a |
| VCL | 70.02 ± 9.47a | 50.07 ± 6.50b | 44.09 ± 4.71b | 41.03 ± 4.05b |
| VSL | 28.39 ± 3.40a | 22.22 ± 2.46b | 19.88 ± 1.85b | 18.81 ± 1.80b |
| VAP | 35.80 ± 4.23a | 27.65 ± 3.02b | 24.84 ± 2.28b | 23.35 ± 2.12b |
| DCL | 15.73 ± 1.82a | 11.34 ± 1.31b | 10.37 ± 1.09b | 9.80 ± 1.01b |
| DSL | 5.18 ± 0.57a | 3.88 ± 0.50b | 3.55 ± 0.45b | 3.39 ± 0.51b |
| DAP | 7.22 ± 0.76a | 5.43 ± 0.63b | 5.00 ± 0.56b | 4.75 ± 0.58b |
| ALH | 0.76 ± 0.09a | 0.56 ± 0.06b | 0.51 ± 0.05b | 0.48 ± 0.04b |
| BCF | 7.71 ± 0.89a | 5.61 ± 0.81b | 4.60 ± 0.67bc | 4.43 ± 0.64c |
| HAC | 0.23 ± 0.02a | 0.21 ± 0.03ab | 0.18 ± 0.01b | 0.18 ± 0.01b |
| WOB | 0.53 ± 0.01b | 0.57 ± 0.01a | 0.57 ± 0.01a | 0.58 ± 0.02a |
| LIN | 0.42 ± 0.02b | 0.46 ± 0.02a | 0.46 ± 0.02a | 0.47 ± 0.02a |
| STR | 0.79 ± 0.01 | 0.80 ± 0.01 | 0.80 ± 0.01 | 0.80 ± 0.01 |
- Abbreviations: ALH, amplitude of lateral head displacement; BCF, beat-cross frequency; DAP, distance average path; DCL, distance curve line; DSL, distance straight line; HAC, head activity; LIN, linearity (VSL/VCL); STR, straightness (VSL/VAP); VAP, average path velocity; VCL, curvilinear velocity; VSL, straight-line velocity; WOB, Wobble (VAP/VCL). a,b,c Values in the row with different letters indicate significant differences (Duncan's multiple-range test).
- a,b,cValues in the row with different letters indicate significant differences (Duncan's multiple-range test).
3.3 The effect of the interaction between breed and extender on sperm quality parameters of cryopreserved goat semen
This experiment was to compare the efficiency of fresh and different levels of lyophilized egg yolk in Tris-base extender with regards to the quality of cryopreserved goat semen in different breeds. In the post-thaw Boer semen mixed with 10%, 15%, and 20% of the lyophilized egg yolk, the results of progressive motility, progressive fast motility, VCL, VSL, VAP, DCL, DSL, DAP, ALH, BCF, and HAC parameters showed significantly different when compared with the control group (p < .05) (Table 3). In the Saanen semen, the parameters of progressive motility and progressive fast motility were significantly lower in the groups supplemented with 10%, 15%, and 20% of lyophilized egg yolk when compared to the control group. However, the parameters of VCL, VSL, VAP, and ALH showed no significant difference between the control group and the group supplemented with 10% of lyophilized egg yolk. (Table 3). Furthermore, in the post-thaw Boer and Saanen semen, the total motility and immotile parameters were no significantly different between the control group and all treatment groups (Table 3). The viability in the group supplemented with 10% of lyophilized egg yolk, was comparable to that in the control group, but supplementing with 15% and 20% of lyophilized egg yolk significantly impaired the sperm viability (Table 3). Next, comparing the quality of cryopreserved semen in Boer and Saanen between the control and treatment groups, we found that there was no any interactions between breed and treatment in the the most of kinetic parameters. However, only HAC and STR showed significant difference between breeds (Boer and Saanen) and treatments (p < .05).
| Parameters | Treatments | |||||||
|---|---|---|---|---|---|---|---|---|
| Control | 10% of lyophilized | 15% of lyophilized | 20% of lyophilized | |||||
| Boer | Saanen | Boer | Saanen | Boer | Saanen | Boer | Saanen | |
| Viability (%) | 66.33 ± 1.47a | 63.67 ± 2.09a | 64.00 ± 1.29a | 60.00 ± 1.61ab | 53.67 ± 2.88bc | 54.67 ± 0.83bc | 53.33 ± 1.32bc | 49.67 ± 2.74c |
| Membrane integrity (%) | 42.00 ± 3.55a | 38.33 ± 0.91ab | 38.67 ± 2.51ab | 33.00 ± 1.44bc | 30.33 ± 1.42bc | 35.67 ± 2.33ab | 27.00 ± 2.46bc | 22.33 ± 1.27c |
| Total motility (%) | 61.80 ± 5.91a | 57.99 ± 2.27ab | 57.45 ± 4.12ab | 49.89 ± 3.29b | 54.70 ± 3.61ab | 49.34 ± 3.78b | 55.33 ± 3.83ab | 48.24 ± 2.83b |
| P motility (%) | 43.85 ± 7.48ab | 51.62 ± 3.21a | 29.16 ± 4.81cd | 38.49 ± 5.02bc | 23.44 ± 3.42d | 34.21 ± 4.89bcd | 22.34 ± 3.84d | 31.01 ± 4.05cd |
| P fast motility (%) | 21.12 ± 4.68a | 20.00 ± 2.48a | 10.29 ± 2.46b | 13.03 ± 2.96b | 7.44 ± 1.37b | 13.07 ± 2.98b | 7.27 ± 1.71b | 8.98 ± 1.68b |
| Immotile (%) | 38.20 ± 5.91b | 42.01 ± 2.27ab | 42.55 ± 4.13ab | 50.11 ± 3.29a | 45.30 ± 3.61ab | 50.66 ± 3.78a | 44.67 ± 3.83ab | 51.82 ± 2.81a |
| VCL | 70.33 ± 10.67a | 69.26 ± 6.01a | 47.50 ± 5.65b | 56.17 ± 8.18ab | 40.88 ± 3.79b | 51.70 ± 6.11b | 39.95 ± 4.16b | 43.60 ± 3.80b |
| VSL | 28.63 ± 3.78a | 27.81 ± 2.38a | 21.00 ± 2.18bc | 24.90 ± 2.99ab | 19.04 ± 1.65bc | 21.86 ± 2.24bc | 17.94 ± 1.70c | 20.88 ± 1.98bc |
| VAP | 35.91 ± 4.75a | 35.54 ± 2.73a | 26.25 ± 2.72bc | 30.99 ± 3.59ab | 23.51 ± 1.98bc | 27.99 ± 2.77bc | 22.46 ± 2.08c | 25.48 ± 2.19bc |
| DCL | 15.67 ± 2.07a | 15.86 ± 1.07a | 11.10 ± 1.40b | 11.93 ± 1.09b | 9.53 ± 0.92b | 12.37 ± 1.30b | 9.58 ± 1.03b | 10.32 ± 1.01b |
| DSL | 5.21 ± 0.64a | 5.11 ± 0.34ab | 3.79 ± 0.54c | 4.09 ± 0.41abc | 3.27 ± 0.44c | 4.24 ± 0.46abc | 3.19 ± 0.43c | 3.88 ± 0.64bc |
| DAP | 7.20 ± 0.87a | 7.28 ± 0.43a | 5.29 ± 0.68b | 5.77 ± 0.48ab | 4.57 ± 0.52b | 6.02 ± 0.58ab | 4.54 ± 0.53b | 5.23 ± 0.68b |
| ALH | 0.75 ± 0.09a | 0.76 ± 0.05a | 0.53 ± 0.06b | 0.62 ± 0.07ab | 0.47 ± 0.04b | 0.60 ± 0.06b | 0.47 ± 0.004b | 0.51 ± 0.04b |
| BCF | 7.65 ± 1.03a | 7.85 ± 0.46a | 5.55 ± 0.91b | 5.75 ± 0.53b | 4.17 ± 0.63b | 5.61 ± 0.69b | 4.18 ± 0.64b | 5.04 ± 0.63b |
| HAC | 0.24 ± 0.03ab | 0.23 ± 0.01abc | 0.19 ± 0.01bcd | 0.25 ± 0.05a | 0.17 ± 0.01d | 0.19 ± 0.02bcd | 0.18 ± 0.01cd | 0.18 ± 0.01cd |
| WOB | 0.53 ± 0.01bc | 0.52 ± 0.02c | 0.56 ± 0.01ab | 0.57 ± 0.02ab | 0.58 ± 0.01a | 0.56 ± 0.02abc | 0.57 ± 0.02ab | 0.59 ± 0.02a |
| LIN | 0.43 ± 0.02bc | 0.40 ± 0.02c | 0.46 ± 0.02ab | 0.46 ± 0.02ab | 0.48 ± 0.02ab | 0.43 ± 0.02bc | 0.46 ± 0.02ab | 0.49 ± 0.03a |
| STR | 0.80 ± 0.01ab | 0.78 ± 0.02b | 0.81 ± 0.01a | 0.80 ± 0.01ab | 0.81 ± 0.01a | 0.78 ± 0.01b | 0.80 ± 0.01ab | 0.82 ± 0.01a |
- Note: Progressive motility (P motility), Progressive fast motility (P fast motility), Curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), distance curve line (DCL), distance straight line (DSL), distance average path (DAP), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF), head activity (HAC), Wobble (WOB = VAP/VCL), linearity (LIN=VSL/VCL), straightness (STR = VSL/VAP). a,b,c,d Values in the row with different letters indicate significant differences (Duncan's multiple-range test).
4 DISCUSSION
Lyophilization or freeze-drying is widely used for the conservation process of biological products, involving both freezing and dehydration to maintain stability for prolonged intervals. Freeze-drying egg yolk has been applied to ram and goat semen extender to solve the limitations of using fresh egg yolk in extender (Alcay et al., 2016; Moustacas et al., 2011). In any case, no additional research was presented with regards to the addition of lyophilized egg yolk in extender for goat semen. We measured viscosity characteristic and density of all treatments. The results showed that control group presented a significant lower (p < .05) kinematic viscosity at 40°C than 10%, 15%, and 20% of the lyophilized egg yolk groups (Figure S1). We found that between addition of 10% and 20% of the lyophilized egg yolk groups the calculated kinematic viscosity due to dense lyophilized egg yolk particle differences between the egg components increases 2.29 times. On the other hand, a density of homogenized extender of control group was not significantly different among the groups supplement with 10%, 15%, and 20% of the lyophilized egg yolk in Tris-base extender (Figure S1). These results were enough to support the data of CASA analysis. These results presented consistently with previous study. Adding different concentrations of egg yolk in the extender can influence the parameters of bull semen viscosity and sperm motility evaluated by a computer-assisted system (Hirai et al., 1997). Our results have shown that there were no significant differences in viability, membrane integrity, total motility, immotile, HAC, and STR parameters in post-thawed goat sperm with 10% additions of lyophilized egg yolk compared with the control group (Table 2). The percentages of progressive motility, progressive fast motility, VCL, VSL, VAP, DCL, DSL, DAP, ALH, and BCF paramenters were significantly (p < .05) lower in the groups supplemented with 10%, 15%, and 20% of lyophilized egg yolk groups when compared with the control group (Table 2). These results may be due to the different of fresh egg yolk and freeze-dried egg yolk composition of extender. Our results were consistent with the other research that described that sperm motility and kinetic parameters present difference when using different extenders (Farrell et al., 1998; Fernandez-Novo et al., 2021; Kumar et al., 2015; Miguel-Jimenez et al., 2020; Naz et al., 2018; Singh et al., 2018). The previous studies used 20% of lyophilized egg yolk based extenders containing various antioxidants on sperm cryopreservation quality in goats. The results showed that the percentage of motility was 48.00 ± 1.07% in the 20% freeze-dried egg yolk–based extenders group and this was consistent with our study (Alcay et al., 2016). Recent study has reported that the cryopreservation of sperm involves many factors such as species that affects sperm quality (Gangawar et al., 2016). The interaction between the two breeds used in this study (Boer and Saanen) was studied in terms of the efficiency of fresh egg yolk and different levels of freeze dried egg yolk in Tris-base extender on the quality of cryopreserved semen. We found that there was no interaction between breed in the most of kinetic parameters such as viability, membrane integrity, total motility, progressive motility, progressive fast motility, immotile, VCL, VSL, VAP, DCL, DSL, DAP, ALH, BCF, WOB, and LIN. However, the interaction between breeds and treatments showed a significant different in terms of HAC and STR parameters in the 10% and 15% of lyophilized egg yolk groups, respectively (Table 3). In conclusion, the lyophilized egg yolk protocol in cryopreserved goat semen showed that the progressive motility and progressive fast motility were lower in the groups supplemented with 10%, 15%, and 20% of lyophilized egg yolk when compared with the control group of Boer and Saanen breeds. However, the lyophilized egg yolk protocol in cryopreserved goat semen presented percentages of total motility to be lower in the 10% lyophilized egg yolk addition group as compared to a control group, but it was a non-significant difference in these two breeds. When 10% of the lyophilized egg yolk was used in the extender this might be an alternative extender for goat semen cryopreservation where an animal's origin represents a microbiological risk. In addition, the lyophilized egg yolk protocol will be applied to cryopreserved goat semen and might be developed to commercial extender in the future.
ACKNOWLEDGEMENTS
We wish to thank the Phukamyao Goat Farm; Phayao, Boonboon Goat Milk Farm; Lampang, Tamfun Sheep and Goat Farm; Phitsanulok, Sai Lom Ngen Goat Farm; Surin, Thailand, for kindly providing animals, facilities and assistance. This study is financially supported by granted proposals from the University of Phayao (RD63100, FF64-UoE010).
CONFLICT OF INTEREST
The authors have no conflicts of interest to declare.
AUTHOR CONTRIBUTIONS
WI and PI designed and performed the experiment. WI, PI, SS, and CL analysed the data. WI drafted the manuscript. WI, Y-JL, PI and TM revised and edited the manuscript.
Open Research
DATA AVAILABILITY
The data that support the findings of this study is available from the authors upon reasonable request.
