Improving Boer Goat Semen Freezing Using Poultry Egg Yolks: A Study in the Mekong Delta of Vietnam

Improving Boer Goat Semen Freezing Using Poultry Egg Yolks: A Study in the Mekong Delta of Vietnam | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Improving Boer Goat Semen Freezing Using Poultry Egg Yolks: A Study in the Mekong Delta of Vietnam Khuong Thi-Thanh Tran, Duy Lam Khanh Nguyen, Nam Van Be Tran, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7037129/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cryopreservation is a widely used method for long-time sperm preservation, supporting the success of artificial insemination program and enhancing livestock genetics. Supplementation of extenders with egg yolk is a common practice for protection of sperm againt harmful effects of cryopreservation. This study aimed to evaluate the effects of different poultry egg yolk types (chicken, duck, and quail) at various concentrations (0, 5, 10, 15, and 20%) on the post-thaw quality of Boer goat sperm. Semen samples collected from four mature Boer male goats at weekly intervals were used. Semen samples were diluted in a Tris-based glycerol extender supplemented with five concentrations of three egg yolk types. The diluted semen was equilibrated at 15°C for 30 minutes, cooled to 5°C for 60 minutes, then exposed to liquid nitrogen vapor for 15 minutes before immersion and storage in liquid nitrogen. After 72 hours, semen was thawed at 37°C for 60 seconds, and sperm quality parameters were assessed. Results indicated that the 15% chicken egg yolk supplementation provided the best sperm preservation (P < 0.05), achieving the highest overall motility (66.69%), progressive motility (54.13%), viability (70.69%), membrane integrity (50.24%), acrosome integrity (80.12%) and the lowest DNA fragmentation rate (13.16%). These findings suggest that 15% chicken egg yolk in a Tris-glycerol extender optimally supports Boer goat sperm preservation. The results offer practical implications for improving artificial insemination success and establishing semen cryobanks for genetic conservation in tropical goat farming systems, particularly in regions such as the Mekong Delta, Vietnam. Artificial insemination Cryopreservation Egg yolk Goat Sperm Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction The success of artificial insemination (AI), genetic advancement, and disease control programs in livestock (Menchaca et al. 2023), all depend on the crucial method of cryopreservation, which enables preservation of semen for long-term in frozen form. However, the freezing and thawing process adversely affects sperm quality through causing oxidative stress, ice crystal formation, and sperm membrane lipid peroxidation, which leads to decreased sperm motility, viability, and fertility (Hai et al. 2024 ). Extensive use of egg yolk-based extenders has been carried out in order to safe guard sperm against such damages (Bustani and Baiee, 2021 ). Particularly, due to the presence of low-density lipoproteins (LDL), which are crucial in stabilizing sperm membranes and lowering oxidative damage during cryopreservation, egg yolk offers vital lipids, phospholipids, and cholesterol for the protection of sperm against harmful effects of cryopreservation (Chang et al. 2025 ). Several poultry egg yolk types, such as chicken (Swelum et al. 2022 ), duck (Wulandari et al. 2020 ), Japanese quail (Maapola et al. 2023 ), pigeon (Akhter et al. 2018 ), turkey and ostrich, have been evaluated for their protective abilities during sperm cryopreservation in various animal species, demonstrating distinct differences in their effectiveness (Chang et al. 2025 ). However, comparative studies particularly looking at the effects of chicken, duck, and quail egg yolks on goat sperm cryopreservation are still limited. It is generally agreed that chicken egg yolk offers better cryoprotection in a number of species. However, there are not many studies that compare this effect across different types of poultry yolk, especially in Boer goats raised in tropical conditions. Different breeds of animals have different levels of semen cryoresistance, and the environment where they live (like temperature and feed quality) can also affect how well they freeze. So, it is still scientifically important to test and confirm how well different egg yolk sources protect Boer goats in order to create protocols that work in specific areas. Boer goat farming in Vietnam, especially in the Mekong Delta area, faces major challenges including high disease incidence and reproductive inefficiencies that seriously limit herd development and economic sustainability (Khuong et al. 2022 ). Efficient cryopreservation techniques using ideal egg yolk extenders can significantly increase reproductive success and support fast recovery and sustainable growth of goat herds. Thus, the purpose of this work was to assess comparative efficacy of various concentrations of chicken, duck and quail egg yolk in maintaining Boer goat sperm quality during cryopreservation. Finding the best egg yolk type and concentration is expected to provide important new perspectives and workable solutions to improve the reproductive performance, genetic advancement, and herd growth in goats kept in the Mekong Delta, Vietnam. 2. Materials and methods 2.1 Animals Four adult Boer male goats, average age 18.25 ± 0.5 months, average weight 48.25 ± 0.65 kg, obtained from experimental animal facility of the Stem Cell Laboratory, Can Tho University, Vietnam were selected for the study. They were fed a diet formulated to meet the nutritional needs of male adult goats as recommended by NRC ( 2007 ). Goats were fed 3 times/day according to rations, and drinking water was fully prepared for goats. The barn area is built to be tall, cool, with a roof, mosquito nets, and clean. The animals underwent routine health checks and vaccinations. 2.2 Preparation of extenders Eggs from chicken, duck, and Japanese quail were obtained from Ba Huan Corporation (Vietnam) and processed under sterile conditions. Yolk was separated, homogenized, and incorporated into a Tris-glycerol extender. Extender was prepared by dissolving 250 mM Tris-hydroxymethylaminomethane (Biobasic, Canada), 88 mM citric acid (Sigma, USA), and 47 mM D-glucose (Thermo Fisher Scientific, USA) in distilled water, adding 80 mg/L Gentamicin (Sigma, USA), and 8% (v/v) glycerol (Thermo Fisher Scientific) as a cryoprotectant. The extenders were stored at 5ºC, the extenders were incubated at 37ºC for 2 hours before use. 2.3 Experimental design Semen was collected from each Boer buck twice weekly (3 ejaculate/goat). Semen samples collected from 4 goats were evaluated for quality and mixed together to ensure sample uniformity. Experiment 1 The semen was diluted with previously prepared extender supplemented with chicken egg yolk (at concentrations of 5, 10, 15 and 20%) at 37°C at an appropriate ratio so that the semen concentration was adjusted to 2 × 10 8 sperm/mL. Experiment 2 The semen was diluted with previously prepared extender supplemented with duck egg yolk (at concentrations of 5, 10, 15 and 20%) at 37°C at an appropriate ratio so that the semen concentration was adjusted to 2 × 10 8 sperm/mL. Experiment 3 The semen was diluted with previously prepared extender supplemented with quail egg yolk (at concentrations of 5, 10, 15 and 20%) at 37°C at an appropriate ratio so that the semen concentration was adjusted to 2 × 10 8 sperm/mL. Diluted semen was loaded into 0.5-mL French straws (at 25°C), equilibrated at 15°C for 30 minutes, cooled to 5°C over 60 minutes, exposed to liquid nitrogen vapor for 15 minutes, and stored in liquid nitrogen. After 72 hours, semen was thawed at 37°C for 60 seconds. Post-thaw sperm quality was assessed for concentration, motility, viability, membrane integrity, acrosome integrity, and DNA fragmentation using standardized protocols, as described below. Then, the effects of the most optimal egg-yolk concentration from Chicken, Duck and Quail were monitored. 2.4 Assessment of post-thaw sperm quality Sperm concentration 10 µL of fresh semen was diluted with 490 µL of NaHCO 3 solution (Sigma, USA) to immobilize the sperm. Then, 9 µL semen sample was put in a counting chamber and examined under a 40× magnification of microscope. Following World Health Organization (WHO, 2021 ), the number of sperm in both counting chambers was counted and sperm concentration was calculated by the formula \(\:\frac{\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{s}\text{p}\text{e}\text{r}\text{m}\:\text{c}\text{o}\text{u}\text{n}\text{t}\:\text{i}\text{n}\:2\:\text{c}\text{o}\text{u}\text{n}\text{t}\text{i}\text{n}\text{g}\:\text{c}\text{h}\text{a}\text{m}\text{b}\text{e}\text{r}\text{s}}{0.8}\times\:{10}^{6}\) (sperm/mL) Sperm motility : 10 µL of sample was placed on a clean glass slide, then a lamen was placed on top, and the sample was viewed under a 40× magnification of microscope. Sperm motility was assessed and classified into 3 types: progressive (swimming and moving over the medium), non-progressive (moving in place), and immotile (not moving). Analyzed were average motility data (Fumuso et al. 2018 ). Sperm viability was determined by the Eosin-Nigrosin staining technique, as described by Boccia et al ( 2007 ). Stained slides were examined under light microscope (400×). The viable sperm did not absorb the Eosin dye and their heads appeared white, while dead sperm absorbed the Eosin stain and appeared red. At least 200 sperm were examined and percentage of viable sperm was calculated. Sperm membrane integrity was determined following incubation of 20 µL semen and 80 µL HOS solution at 37°C for 30 minutes to allow the hypo-osmotic swelling test (HOST) to be conducted. Following incubation, a slide was prepared and examined under light microscope (400×). Whereas sperm with damaged plasma membrane showed no swelling, sperm with intact membranes showed swelling or curling of the tail (Tran et al. 2025 ). At least 200 sperm were examined and percentage of sperm with intact plasma membrane was calculated. Sperm acrosome integrity was estimated after staining the semen with Giemsa stain (Sigma, USA). The stained slides were examined under light microscope (400×). Sperm with intact acrosomes revealed a pink-stained pole region while sperm with aberrant acrosomes did not absorb the dye (Ministry of Health of Vietnam, 2016 ). The Sperm Chromatin Dispersion (SCD) method was used to evaluate DNA fragmentation, as described previously (Absalan et al. 2012 ). The SCD relies on the principle that sperm with intact DNA will form a characteristic “halo” (a circle of light in the head of sperm) of dispersed DNA loops after denaturation and protein removal, while sperm with fragmented DNA will show small of no halos in the nucleus. 2.5 Statistical analysis Statistical analysis was conducted to examine the impact of egg yolk concentration on semen quality parameters. One-way ANOVA was employed to analyze the data, after confirming normality and homogeneity of variance. Mean comparisons between treatments were conducted using the Tukey method in R software (version 4.3.1; R Development Core Team; New Zealand). the fixed effect was egg yolk concentrations, and the random effects were goats and ejaculations. The results are presented as mean ± the standard deviation (Sd). Statistical significance was set at p < 0.05, indicating a high level of confidence in the obtained results. 3. Results Our results showed that the different concentrations of chicken egg yolk significantly improved sperm quality parameters compared to control (P < 0.05). In general, sperm quality parameters improved as the chicken egg yolk concentration in extender was increased up to 15%, followed by a decline in semen quality with 20% chicken egg yolk (Fig. 2 ). All sperm quality parameters in the extender supplemented with 15% chicken egg yolk were the best and statistically better compared to the other treatment groups (P < 0.05). Specifically, the overall motility, progressive motility rate, acrosome integrity, viability, DNA fragmentation, and membrane integrity rate of sperm in the extender supplemented with 15% chicken egg yolk (C15) were: 66.69 ± 0.60, 54.13 ± 0.46, 80.12 ± 1.15, 70.69 ± 0.61, 13.16 ± 0.93 and 50.24 ± 0.37%, respectively. When the duck egg yolk was considered, the results were quite similar to those of chicken egg yolk (Fig. 3 ). It was observed that the Tris-citrate-glycerol extender containing 15% duck egg yolk had the best sperm quality metrics and was significantly different from the other treatments (P < 0.05). Sperm samples preserved in extender containing 15% duck egg yolk (D15) had the following overall motility, progressive motility, acrosome integrity, viability, DNA fragmentation, and membrane integrity rate: 62.04 ± 0.97, 51.34 ± 0.49, 69.72 ± 1.01, 66.04 ± 0.97, 14.01 ± 0.99% and 42.73 ± 0.73%, respectively (Fig. 3 ). Similar to chicken and duck egg yolk, quail egg yolk also showed the ability to protect goat sperm against the adverse effects of cryoprotection (Fig. 4 ). Tris-citrate-glycerol extender supplemented with 15% quail egg yolk also showed the best results in terms of post-thaw sperm quality, the difference with the other concentrations was statistically significant (P < 0.05). Specifically, the overall sperm motility, progressive motility, acrosome integrity, viability, DNA fragmentation, and membrane integrity rate of sperm were: 57.62 ± 1.14, 47.93 ± 1.01, 68.78 ± 1.03, 61.62 ± 1,14, 14.62 ± 1.11 and 41.93 ± 0.57%, respectively. Table 1 Results of sperm quality analysis in 3 types of egg yolk at 15% concentration each (Chicken 15%-C15; Duck 15%-D15 and Quail 15%-Q15) Overall motility (%) Progressive motility (%) Viability (%) Membrane integrity (%) Acrosome integrity (%) DNA fragmentation (%) C15 66.69 ± 0.59 a 54.13 ± 0.37 a 70.69 ± 0.58 a 50.24 ± 0.89 a 80.12 ± 0.85 a 13.16 ± 0.24 a D15 62.04 ± 0.77 b 51.33 ± 0.49 b 66.04 ± 0.79 b 42.73 ± 0.73 b 69.72 ± 0.87 b 14.01 ± 0.27 b Q15 57.62 ± 0.73 c 47.93 ± 0.36 c 61.62 ± 0.64 c 41.93 ± 0.71 b 68.78 ± 0.76 b 14.62 ± 0.48 b Values with different superscripts within a column are significantly different (P < 0.05). In the present study, supplement of Tris-citrate-glycerol extender with 15% egg yolk from chicken, duck or quail showed best results in terms of post-thaw quality of goat sperm. Therefore, 15% egg yolk supplementation of each type was used for comparison among three egg yolk types. The results presented showed that chicken egg yolk shows the highest values in terms of overall sperm motility and progressive motility, followed by duck egg yolk, while quail egg yolk yielded the lowest results, the difference among three egg yolk types was significant (P < 0.05). Similarly, 15% chicken egg yolk showed better results in terms of sperm viability, membrane integrity, acrosome integrity and DNA integrity compared to duck and quail egg yolk (P < 0.05); however, the difference in these parameters between duck and quail egg yolks was non-significant (Table 1 ). 4. Discussion The present study demonstrates that the type and concentration of egg yolk in the extender significantly affect the cryosurvival of Boer goat sperm. Our results indicate that supplementation of Tris-citrate-glycerol extender with 15% chicken egg yolk provides significantly superior post-thaw sperm quality manifested by higher sperm motility, viability, membrane integrity and acrosome integrity, as well as lower levels of DNA fragmentation when compared with equivalent concentrations of duck or quail egg yolk. These results are supported by the findings of some previous studies including Benchaib et al ( 2003 ) and Akhter et al ( 2018 ). On the other hand, extenders without egg yolk constantly produced poor sperm quality, which emphasizes the indispensable function of egg yolk components in protecting spermatozoa during cryopreservation (Donnelly et al. 2001 ). There was a clear dose-dependent effect of egg yolk on post-thaw quality of goat sperm; supplementation of Tris-citrate-glycerol extender with 15% egg yolk was ideal, even though 5% and 10% egg yolk supplementation also showed some protective effect. Thus, 15% egg yolk concentration seems to provides a critical threshold of cryoprotective agents including low-density lipoproteins (LDL), cholesterol, and intrinsic antioxidants required to preserve the structural and functional integrity of the sperm membrane (Marri and Richner, 2014 ; Sen et al. 2015 ). On the other hand, it appeared that raising the egg yolk concentration to 20% showed significantly poor results compared to those obtained by 15% egg yolk supplementation. This is most likely due to increased yolk aggregates forming, which raise extender viscosity, impede sperm motility, and may cause strong membrane stabilization, preventing the post-thaw sperm from returning to normal physiological state (Blanch et al. 2014 ; Tarig et al. 2017 ). Forming a protective barrier around sperm cells, egg yolk functions as a non-permeating cryoprotectant. Particularly important is its LDL fraction, since it reacts with the sperm plasma membrane to add necessary phospholipids and cholesterol, minimizing cold shock effects and lowering the risk of membrane rupture during freezing and thawing (Akhter et al. 2018 ). Moreover, egg yolk contains antioxidants including vitamin E, selenium, and carotenoids that scavenge reactive oxygen species produced during cryopreservation, so preserving both membrane integrity and DNA structure of sperm (Zhong and Zhou, 2013 ). Our results clearly show that chicken egg yolk confers better cryoprotection to goat sperm than duck and quail egg yolk when compared in terms of post-thaw sperm quality parameters. Relatively better post-thaw sperm quality parameters recorded following 15% egg yolk supplementation can be attributed in part to its higher polyunsaturated fatty acids and antioxidant vitamin contents compared to duck or quail egg yolk. These biochemical characteristics are known to explain more efficient oxidative protection and membrane stabilization (Akhter et al. 2018 ; Wulandari et al. 2020 ). Duck egg yolk showed an intermediate position; the rather higher water content and larger proportion of saturated fats in quail egg yolk may lead to a lower concentration of protective solids. This hierarchy emphasizes the species-specific interaction between the requirements of the sperm membrane and the biochemical composition of egg yolk (Menchaca, 2023 ). The results of this study have practical relevance with the Boer goat farming in the Mekong Delta of Vietnam. High disease incidence and reproductive problems that hinder herd development and economic sustainability (Khuong et al. 2022 ) present serious problems for the goat farmers of this area. Veterinary practitioners and goat breeders can greatly improve semen quality of their animals by using a cryopreservation technique including the use of Tris-citrate-glycerol extender supplemented with 15% chicken egg yolk, ensuring the success of artificial insemination program and enabling genetic improvement and herd expansion. Crucially for the preservation of valuable species and for reducing losses during disease outbreaks or other adverse conditions, this optimized protocol also supports the building of genetic resource banks (Khuong et al. 2022 ). Moreover, our work prepares the way for future studies aimed to separate the specific protective fractions within chicken egg yolk, such as purified LDL and assess the possible advantages of use of additional antioxidant supplements. These improvements might help in further improving in vitro sperm quality and in vivo fertility results in goats(Blanch et al. 2014 ; Tarig et al. 2017 ). Overall, our results show that a Tris-citrate-glycerol extender supplemented with 15% chicken egg yolk best preserves Boer goat sperm by properly balancing membrane stabilization with antioxidant defense. These findings not only deepen our knowledge of sperm cryobiology but also provide a workable and scientifically proved solution to increase herd sustainability and reproductive efficiency in areas where Boer goat farming is of great economic importance. 5. Conclusion The results of the present study show that adding 15% chicken egg yolk to a Tris-citrate-glycerol extender greatly improves the post-thaw quality of frozen Boer goat semen compared to egg yolk from ducks and quails. Especially, compared to other treatments, the extender supplemented with 15% chicken egg yolk resulted in better post-thaw sperm motility, viability, membrane integrity, acrosome integrity and DNA fragmentation control. These findings highlight the possibility of this formulation to enhance cryopreservation results and, hence, the reproductive efficiency in Boer goat breeding projects. Declarations Animal ethics statement Ethical approval was obtained for the animal care, housing, and semen collection procedures, following the guidelines of the Regulation on Ethics in Animal Experimentation of Can Tho University (CTU Animal Ethics Committee 2024, code CTU-AEC24013). Acknowledgements We thank the staff at the Stem Cell Laboratory, Can Tho University for their support and assistance during the research process. Funding This study was financially supported by the Ministry of Education and Training, Vietnam, Code: B2024-TCT-04. Author Contributions Khuong Thi Thanh Tran contributed to Methodology and took the lead in Reviewing and Editing the manuscript. Duy Lam Khanh Nguyen was responsible for Software, Validation, and led the Writing of the Original Draft. Nam Van Be Tran contributed to Conceptualization, Data Curation and Visualization. Giang Thi Tran was responsible for Investigation and Supervision, Competing Interests The authors have no relevant financial or non-financial interests to disclose. Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request Consent to publish All authors consented to publication of the study. References Absalan F, Ghannadi A, Kazerooni M, Parifar R, Jamalzadeh F, Amiri S (2012) Value of sperm chromatin dispersion test in couples with unexplained recurrent abortion. J Assist Reprod Genet 29(1):11–14. https://doi.org/10.1007/s10815-011-9647-0 Akhter S, Rakha BA, Ansari MS, Iqbal S, Khalid M (2018) Evaluation of pigeon egg yolk for post–thaw quality, enzyme leakage and fertility of buffalo (Bubalus bubalis) bull spermatozoa. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7037129","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":483049821,"identity":"26d3323f-1418-4e33-bdb0-4ccf2254ece7","order_by":0,"name":"Khuong Thi-Thanh Tran","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzklEQVRIiWNgGAWjYHACZmYwxd4AphgbCKnnYWOGauE5AFZNihaJBCK12Mv3HzYubLPJk498/PwxD4ON7IYDPGYPCNmSPLMtrdjwdpphMw9DmjFQi7kBIS2HedsOJ26cncMI1HI4ccMBtjQJ4rTMPAPS8p84LckgLfMleEBaDgC1MB/Dr+VYsrExz7m0xA08aYYz5xgkG888TEALe/PBx9I8ZTaJ89sPP/jwpsJOtu94YxteLXBgcABMAjEzUeqBQL6BWJWjYBSMglEw4gAAYpZCoDBmRuMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0000-8145-9822","institution":"Can Tho University","correspondingAuthor":true,"prefix":"","firstName":"Khuong","middleName":"Thi-Thanh","lastName":"Tran","suffix":""},{"id":483049822,"identity":"1baace31-d84f-45cf-824e-0d650c408111","order_by":1,"name":"Duy Lam Khanh Nguyen","email":"","orcid":"","institution":"Can Tho University","correspondingAuthor":false,"prefix":"","firstName":"Duy","middleName":"Lam Khanh","lastName":"Nguyen","suffix":""},{"id":483049823,"identity":"d87bde9c-dd21-4e53-9a22-79be920e8dc0","order_by":2,"name":"Nam Van Be Tran","email":"","orcid":"","institution":"Can Tho University","correspondingAuthor":false,"prefix":"","firstName":"Nam","middleName":"Van Be","lastName":"Tran","suffix":""},{"id":483049824,"identity":"e9764e21-185e-483e-a746-59be385967c3","order_by":3,"name":"Giang Thi Tran","email":"","orcid":"","institution":"Can Tho University","correspondingAuthor":false,"prefix":"","firstName":"Giang","middleName":"Thi","lastName":"Tran","suffix":""}],"badges":[],"createdAt":"2025-07-03 10:39:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7037129/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7037129/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":86787226,"identity":"e5f2008c-ceb7-4f03-b7f7-2b13b8675a21","added_by":"auto","created_at":"2025-07-15 14:17:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":176724,"visible":true,"origin":"","legend":"\u003cp\u003eSperm quality test\u003c/p\u003e\n\u003cp\u003eA - Sperm stained with eosin-nigrosin; Live sperm do not take up the dye (red arrow), dead sperm take up the dye (black arrow). B - Sperm tested for HOS; Sperm with intact cell membranes show tail curling reaction (red arrow), sperm with damaged cell membranes do not have reaction (black arrow). C - Sperm tested for acrosome integrity; Sperm with intact acrosome take up the dye (red arrow), sperm with damaged acrosome do not take up the dye (black arrow). D - Sperm tested for DNA fragmentation; Sperm with halo has intact DNA (red arrow), sperm without halo has fragmented DNA (black arrow). Scale bar = 50µm.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7037129/v1/4b3d7f114f78228470e33122.png"},{"id":86786375,"identity":"163f2d71-b13c-44b2-92ce-a889c4ede8c2","added_by":"auto","created_at":"2025-07-15 14:09:43","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":295695,"visible":true,"origin":"","legend":"\u003cp\u003eGoat sperm quality after cryopreservation with Tris-citrate-glycerol extender supplemented with different concentrations of chicken egg yolk. Values for each parameter with different superscripts are significantly different (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7037129/v1/b28a711bf3215fad4f7ac7bd.jpeg"},{"id":86787229,"identity":"11bf7481-3abe-4859-a22d-94fd5a0a5c17","added_by":"auto","created_at":"2025-07-15 14:17:43","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":299682,"visible":true,"origin":"","legend":"\u003cp\u003eGoat sperm quality after cryopreservation with Tris-citrate-glycerol extender supplemented with different concentrations of duck egg yolk. Values for each parameter with different superscripts are significantly different (P\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7037129/v1/096678c05475277450af27ad.jpeg"},{"id":86786383,"identity":"bb231c99-ec40-433a-b829-cf827cce76d8","added_by":"auto","created_at":"2025-07-15 14:09:43","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":301177,"visible":true,"origin":"","legend":"\u003cp\u003eGoat sperm quality after cryopreservation with Tris-citrate-glycerol extender supplemented with different concentrations of quail egg yolk. Values for each parameter with different superscripts are significantly different (P\u0026lt;0.05)\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7037129/v1/d3a143e564499d20a34bc5c1.jpeg"},{"id":90427391,"identity":"cffaefb0-54ad-4fec-a028-ca169872e5bd","added_by":"auto","created_at":"2025-09-02 14:57:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1633341,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7037129/v1/5ce48872-04d8-4647-a383-b94e3ae9d129.pdf"}],"financialInterests":"","formattedTitle":"Improving Boer Goat Semen Freezing Using Poultry Egg Yolks: A Study in the Mekong Delta of Vietnam","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe success of artificial insemination (AI), genetic advancement, and disease control programs in livestock (Menchaca et al. 2023), all depend on the crucial method of cryopreservation, which enables preservation of semen for long-term in frozen form. However, the freezing and thawing process adversely affects sperm quality through causing oxidative stress, ice crystal formation, and sperm membrane lipid peroxidation, which leads to decreased sperm motility, viability,\u0026ensp;and fertility (Hai et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eExtensive use of egg yolk-based extenders has been carried out in order to safe guard sperm against such damages (Bustani and Baiee, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Particularly, due to the presence of low-density lipoproteins (LDL), which are crucial in stabilizing sperm membranes and lowering oxidative damage during cryopreservation, egg yolk offers vital lipids, phospholipids, and cholesterol for the protection of sperm against harmful effects of cryopreservation (Chang et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSeveral poultry egg yolk types, such as chicken (Swelum et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), duck (Wulandari et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), Japanese quail (Maapola et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), pigeon (Akhter et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), turkey and ostrich, have been evaluated for their protective abilities during sperm cryopreservation in various animal species, demonstrating distinct differences in their effectiveness (Chang et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, comparative studies particularly looking at the effects of chicken, duck, and quail egg yolks on goat sperm cryopreservation are still limited.\u003c/p\u003e\u003cp\u003eIt is generally agreed that chicken egg yolk offers better cryoprotection in a number of species. However, there are not many studies that compare this effect across different types of poultry yolk, especially in Boer goats raised in tropical conditions. Different breeds of animals have different levels of semen cryoresistance, and the environment where they live (like temperature and feed quality) can also affect how well they freeze. So, it is still scientifically important to test and confirm how well different egg yolk sources protect Boer goats in order to create protocols that work in specific areas. Boer goat farming in Vietnam, especially in the Mekong Delta area, faces major challenges including high disease incidence and reproductive inefficiencies that seriously limit herd development and economic sustainability (Khuong et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Efficient cryopreservation techniques using ideal egg yolk extenders can significantly increase reproductive success and support fast recovery and sustainable growth of goat herds. Thus, the purpose of this work was to assess comparative efficacy of various concentrations of chicken, duck and quail egg yolk in maintaining Boer goat sperm quality during cryopreservation. Finding the best egg yolk type and concentration is expected to provide important new perspectives and workable solutions to improve the reproductive performance, genetic advancement, and herd growth in goats kept in the Mekong Delta, Vietnam.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Animals\u003c/h2\u003e\u003cp\u003e Four adult Boer male goats, average age 18.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 months, average weight 48.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65 kg, obtained from experimental animal facility of the Stem Cell Laboratory, Can Tho University, Vietnam were selected for the study. They were fed a diet formulated to meet the nutritional needs of male adult goats as recommended by NRC (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Goats were fed 3 times/day according to rations, and drinking water was fully prepared for goats. The barn area is built to be tall, cool, with a roof, mosquito nets, and clean. The animals underwent routine health checks and vaccinations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Preparation of extenders\u003c/h2\u003e\u003cp\u003eEggs from chicken, duck, and Japanese quail were obtained from Ba Huan Corporation (Vietnam) and processed under sterile conditions. Yolk was separated, homogenized, and incorporated into a Tris-glycerol extender. Extender was prepared by dissolving 250 mM Tris-hydroxymethylaminomethane (Biobasic, Canada), 88 mM citric acid (Sigma, USA), and 47 mM D-glucose (Thermo Fisher Scientific, USA) in distilled water, adding 80 mg/L Gentamicin (Sigma, USA), and 8% (v/v) glycerol (Thermo Fisher Scientific) as a cryoprotectant. The extenders were stored at 5\u0026ordm;C, the extenders were incubated at 37\u0026ordm;C for 2 hours before use.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Experimental design\u003c/h2\u003e\u003cp\u003eSemen was collected from each Boer buck twice weekly (3 ejaculate/goat). Semen samples collected from 4 goats were evaluated for quality and mixed together to ensure sample uniformity.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eExperiment 1\u003c/strong\u003e\u003cp\u003eThe semen was diluted with previously prepared extender supplemented with chicken egg yolk (at concentrations of 5, 10, 15 and 20%) at 37\u0026deg;C at an appropriate ratio so that the semen concentration was adjusted to 2 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e sperm/mL.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eExperiment 2\u003c/strong\u003e\u003cp\u003eThe semen was diluted with previously prepared extender supplemented with duck egg yolk (at concentrations of 5, 10, 15 and 20%) at 37\u0026deg;C at an appropriate ratio so that the semen concentration was adjusted to 2 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e sperm/mL.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eExperiment 3\u003c/strong\u003e\u003cp\u003eThe semen was diluted with previously prepared extender supplemented with quail egg yolk (at concentrations of 5, 10, 15 and 20%) at 37\u0026deg;C at an appropriate ratio so that the semen concentration was adjusted to 2 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e sperm/mL.\u003c/p\u003e\u003c/p\u003e\u003cp\u003eDiluted semen was loaded into 0.5-mL French straws (at 25\u0026deg;C), equilibrated at 15\u0026deg;C for 30 minutes, cooled to 5\u0026deg;C over 60 minutes, exposed to liquid nitrogen vapor for 15 minutes, and stored in liquid nitrogen. After 72 hours, semen was thawed at 37\u0026deg;C for 60 seconds. Post-thaw sperm quality was assessed for concentration, motility, viability, membrane integrity, acrosome integrity, and DNA fragmentation using standardized protocols, as described below. Then, the effects of the most optimal egg-yolk concentration from Chicken, Duck and Quail were monitored.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Assessment of post-thaw sperm quality\u003c/h2\u003e\u003cp\u003e\u003cstrong\u003eSperm concentration\u003c/strong\u003e\u003cp\u003e10 \u0026micro;L of fresh semen was diluted with 490 \u0026micro;L of NaHCO\u003csub\u003e3\u003c/sub\u003e solution (Sigma, USA) to immobilize the sperm. Then, 9 \u0026micro;L semen sample was put in a counting chamber and examined under a 40\u0026times; magnification of microscope. Following World Health Organization (WHO, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), the number of sperm in both counting chambers was counted and sperm concentration was calculated by the formula\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{T}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{s}\\text{p}\\text{e}\\text{r}\\text{m}\\:\\text{c}\\text{o}\\text{u}\\text{n}\\text{t}\\:\\text{i}\\text{n}\\:2\\:\\text{c}\\text{o}\\text{u}\\text{n}\\text{t}\\text{i}\\text{n}\\text{g}\\:\\text{c}\\text{h}\\text{a}\\text{m}\\text{b}\\text{e}\\text{r}\\text{s}}{0.8}\\times\\:{10}^{6}\\)\u003c/span\u003e\u003c/span\u003e (sperm/mL)\u003c/p\u003e\u003cp\u003e\u003cem\u003eSperm motility\u003c/em\u003e: 10 \u0026micro;L of sample was placed on a clean glass slide, then a lamen was placed on top, and the sample was viewed under a 40\u0026times; magnification of microscope. Sperm motility was assessed and classified into 3 types: progressive (swimming and moving over the medium), non-progressive (moving in place), and immotile (not moving). Analyzed were average motility data (Fumuso et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eSperm viability\u003c/em\u003e was determined by the Eosin-Nigrosin staining technique, as described by Boccia et al (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Stained slides were examined under light microscope (400\u0026times;). The viable sperm did not absorb the Eosin dye and their heads appeared white, while dead sperm absorbed the Eosin stain and appeared red. At least 200 sperm were examined and percentage of viable sperm was calculated.\u003c/p\u003e\u003cp\u003e\u003cem\u003eSperm membrane integrity\u003c/em\u003e was determined following incubation of 20 \u0026micro;L semen and 80 \u0026micro;L HOS solution at 37\u0026deg;C for 30 minutes to allow the hypo-osmotic swelling test (HOST) to be conducted. Following incubation, a slide was prepared and examined under light microscope (400\u0026times;). Whereas sperm with damaged plasma membrane showed no swelling, sperm with intact membranes showed swelling or curling of the tail (Tran et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). At least 200 sperm were examined and percentage of sperm with intact plasma membrane was calculated.\u003c/p\u003e\u003cp\u003e\u003cem\u003eSperm acrosome integrity\u003c/em\u003e was estimated after staining the semen with Giemsa stain (Sigma, USA). The stained slides were examined under light microscope (400\u0026times;). Sperm with intact acrosomes revealed a pink-stained pole region while sperm with aberrant acrosomes did not absorb the dye (Ministry of Health of Vietnam, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eThe Sperm Chromatin Dispersion\u003c/em\u003e (SCD) method was used to evaluate DNA fragmentation, as described previously (Absalan et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The SCD relies on the principle that sperm with intact DNA will form a characteristic \u0026ldquo;halo\u0026rdquo; (a circle of light in the head of sperm) of dispersed DNA loops after denaturation and protein removal, while sperm with fragmented DNA will show small of no halos in the nucleus.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Statistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analysis was conducted to examine the impact of egg yolk concentration on semen quality parameters. One-way ANOVA was employed to analyze the data, after confirming normality and homogeneity of variance. Mean comparisons between treatments were conducted using the Tukey method in R software (version 4.3.1; R Development Core Team; New Zealand). the fixed effect was egg yolk concentrations, and the random effects were goats and ejaculations. The results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;the standard deviation (Sd). Statistical significance was set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, indicating a high level of confidence in the obtained results.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eOur results showed that the different concentrations of chicken egg yolk significantly improved sperm quality parameters compared to control (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In general, sperm quality parameters improved as the chicken egg yolk concentration in extender was increased up to 15%, followed by a decline in semen quality with 20% chicken egg yolk (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). All sperm quality parameters in the extender supplemented with 15% chicken egg yolk were the best and statistically better compared to the other treatment groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Specifically, the overall motility, progressive motility rate, acrosome integrity, viability, DNA fragmentation, and membrane integrity rate of sperm in the extender supplemented with 15% chicken egg yolk (C15) were: 66.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60, 54.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46, 80.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.15, 70.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.61, 13.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93 and 50.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37%, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWhen the duck egg yolk was considered, the results were quite similar to those of chicken egg yolk (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). It was observed that the Tris-citrate-glycerol extender containing 15% duck egg yolk had the best sperm quality metrics and was significantly different from the other treatments (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Sperm samples preserved in extender containing 15% duck egg yolk (D15) had the following overall motility, progressive motility, acrosome integrity, viability, DNA fragmentation, and membrane integrity rate: 62.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97, 51.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49, 69.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01, 66.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97, 14.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99% and 42.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSimilar to chicken and duck egg yolk, quail egg yolk also showed the ability to protect goat sperm against the adverse effects of cryoprotection (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Tris-citrate-glycerol extender supplemented with 15% quail egg yolk also showed the best results in terms of post-thaw sperm quality, the difference with the other concentrations was statistically significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Specifically, the overall sperm motility, progressive motility, acrosome integrity, viability, DNA fragmentation, and membrane integrity rate of sperm were: 57.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.14, 47.93\u0026thinsp;\u0026plusmn;\u0026thinsp;1.01, 68.78\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03, 61.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1,14, 14.62\u0026thinsp;\u0026plusmn;\u0026thinsp;1.11 and 41.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57%, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eResults of sperm quality analysis in 3 types of egg yolk at 15% concentration each (Chicken 15%-C15; Duck 15%-D15 and Quail 15%-Q15)\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eOverall motility (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eProgressive motility (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eViability\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMembrane integrity (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eAcrosome integrity\u003c/p\u003e\u003cp\u003e(%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eDNA fragmentation (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eC15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e66.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e54.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e70.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e50.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e80.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e13.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eD15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e62.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e51.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e66.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e42.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e69.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e14.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eQ15\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e57.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e47.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e61.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e41.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e68.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e14.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eValues with different superscripts within a column are significantly different (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003eIn the present study, supplement of Tris-citrate-glycerol extender with 15% egg yolk from chicken, duck or quail showed best results in terms of post-thaw quality of goat sperm. Therefore, 15% egg yolk supplementation of each type was used for comparison among three egg yolk types. The results presented showed that chicken egg yolk shows the highest values in terms of overall sperm motility and progressive motility, followed by duck egg yolk, while quail egg yolk yielded the lowest results, the difference among three egg yolk types was significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, 15% chicken egg yolk showed better results in terms of sperm viability, membrane integrity, acrosome integrity and DNA integrity compared to duck and quail egg yolk (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05); however, the difference in these parameters between duck and quail egg yolks was non-significant (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe present study demonstrates that the type and concentration of egg yolk in the extender significantly affect the cryosurvival of Boer goat sperm. Our results indicate that supplementation of Tris-citrate-glycerol extender with 15% chicken egg yolk provides significantly superior post-thaw sperm quality manifested by higher sperm motility, viability, membrane integrity and acrosome integrity, as well as lower levels of DNA fragmentation when compared with equivalent concentrations of duck or quail egg yolk. These results are supported by the findings of some previous studies including Benchaib et al (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) and Akhter et al (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). On the other hand, extenders without egg yolk constantly produced poor sperm quality, which emphasizes the indispensable function of egg yolk components in protecting spermatozoa during cryopreservation (Donnelly et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThere was a clear dose-dependent effect of egg yolk on post-thaw quality of goat sperm; supplementation of Tris-citrate-glycerol extender with 15% egg yolk was ideal, even though 5% and 10% egg yolk supplementation also showed some protective effect. Thus, 15% egg yolk concentration seems to provides a critical threshold of cryoprotective agents including low-density lipoproteins (LDL), cholesterol, and intrinsic antioxidants required to preserve the structural and functional integrity of the sperm membrane (Marri and Richner, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Sen et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). On the other hand, it appeared that raising the egg yolk concentration to 20% showed significantly poor results compared to those obtained by 15% egg yolk supplementation. This is most likely due to increased yolk aggregates forming, which raise extender viscosity, impede sperm motility, and may cause strong membrane stabilization, preventing the post-thaw sperm from returning to normal physiological state (Blanch et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Tarig et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eForming a protective barrier around sperm cells, egg yolk functions as a non-permeating cryoprotectant. Particularly important is its LDL fraction, since it reacts with the sperm plasma membrane to add necessary phospholipids and cholesterol, minimizing cold shock effects and lowering the risk of membrane rupture during freezing and thawing (Akhter et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Moreover, egg yolk contains antioxidants including vitamin E, selenium, and carotenoids that scavenge reactive oxygen species produced during cryopreservation, so preserving both membrane integrity and DNA structure of sperm (Zhong and Zhou, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOur results clearly show that chicken egg yolk confers better cryoprotection to goat sperm than duck and quail egg yolk when compared in terms of post-thaw sperm quality parameters. Relatively better post-thaw sperm quality parameters recorded following 15% egg yolk supplementation can be attributed in part to its higher polyunsaturated fatty acids and antioxidant vitamin contents compared to duck or quail egg yolk. These biochemical characteristics are known to explain more efficient oxidative protection and membrane stabilization (Akhter et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wulandari et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Duck egg yolk showed an intermediate position; the rather higher water content and larger proportion of saturated fats in quail egg yolk may lead to a lower concentration of protective solids. This hierarchy emphasizes the species-specific interaction between the requirements of the sperm membrane and the biochemical composition of egg yolk (Menchaca, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe results of this study have practical relevance with the Boer goat farming in the Mekong Delta of Vietnam. High disease incidence and reproductive problems that hinder herd development and economic sustainability (Khuong et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) present serious problems for the goat farmers of this area. Veterinary practitioners and goat breeders can greatly improve semen quality of their animals by using a cryopreservation technique including the use of Tris-citrate-glycerol extender supplemented with 15% chicken egg yolk, ensuring the success of artificial insemination program and enabling genetic improvement and herd expansion. Crucially for the preservation of valuable species and for reducing losses during disease outbreaks or other adverse conditions, this optimized protocol also supports the building of genetic resource banks (Khuong et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMoreover, our work prepares the way for future studies aimed to separate the specific protective fractions within chicken egg yolk, such as purified LDL and assess the possible advantages of use of additional antioxidant supplements. These improvements might help in further improving in vitro sperm quality and in vivo fertility results in goats(Blanch et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Tarig et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eOverall, our results show that a Tris-citrate-glycerol extender supplemented with 15% chicken egg yolk best preserves Boer goat sperm by properly balancing membrane stabilization with antioxidant defense. These findings not only deepen our knowledge of sperm cryobiology but also provide a workable and scientifically proved solution to increase herd sustainability and reproductive efficiency in areas where Boer goat farming is of great economic importance.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe results of the present study show that adding 15% chicken egg yolk to a Tris-citrate-glycerol extender greatly improves the post-thaw quality of frozen Boer goat semen compared to egg yolk from ducks and quails. Especially, compared to other treatments, the extender supplemented with 15% chicken egg yolk resulted in better post-thaw sperm motility, viability, membrane integrity, acrosome integrity and DNA fragmentation control. These findings highlight the possibility of this formulation to enhance cryopreservation results and, hence, the reproductive efficiency in Boer goat breeding projects.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAnimal ethics statement\u0026nbsp;\u003c/strong\u003eEthical approval was obtained for the animal care, housing, and semen collection procedures, following the guidelines of the Regulation on Ethics in Animal Experimentation of Can Tho University (CTU Animal Ethics Committee 2024, code CTU-AEC24013).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eWe thank the staff at the Stem Cell Laboratory, Can Tho University for their support and assistance during the research process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis study was \u0026nbsp;financially \u0026nbsp; supported \u0026nbsp;by \u0026nbsp;the Ministry \u0026nbsp; of \u0026nbsp;Education \u0026nbsp;and \u0026nbsp; Training, \u0026nbsp;Vietnam, Code: B2024-TCT-04.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003eKhuong Thi Thanh Tran contributed to Methodology and took the lead in Reviewing and Editing the manuscript. Duy Lam Khanh Nguyen was responsible for Software, Validation, and led the Writing of the Original Draft. Nam Van Be Tran contributed to Conceptualization, Data Curation and Visualization. Giang Thi Tran was responsible for Investigation and Supervision,\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to publish\u003c/strong\u003e All authors consented to publication of the study.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbsalan F, Ghannadi A, Kazerooni M, Parifar R, Jamalzadeh F, Amiri S (2012) Value of sperm chromatin dispersion test in couples with unexplained recurrent abortion. 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J Integr Agric 12(10):1826\u0026ndash;1838. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S2095-3119(13)60412-8\u003c/span\u003e\u003cspan address=\"10.1016/S2095-3119(13)60412-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Artificial insemination, Cryopreservation, Egg yolk, Goat, Sperm","lastPublishedDoi":"10.21203/rs.3.rs-7037129/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7037129/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCryopreservation is a widely used method for long-time sperm preservation, supporting the success of artificial insemination program and enhancing livestock genetics. Supplementation of extenders with egg yolk is a common practice for protection of sperm againt harmful effects of cryopreservation. This study aimed to evaluate the effects of different poultry egg yolk types (chicken, duck, and quail) at various concentrations (0, 5, 10, 15, and 20%) on the post-thaw quality of Boer goat sperm. Semen samples collected from four mature Boer male goats at weekly intervals were used. Semen samples were diluted in a Tris-based glycerol extender supplemented with five concentrations of three egg yolk types. The diluted semen was equilibrated at 15\u0026deg;C for 30 minutes, cooled to 5\u0026deg;C for 60 minutes, then exposed to liquid nitrogen vapor for 15 minutes before immersion and storage in liquid nitrogen. After 72 hours, semen was thawed at 37\u0026deg;C for 60 seconds, and sperm quality parameters were assessed. Results indicated that the 15% chicken egg yolk supplementation provided the best sperm preservation (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), achieving the highest overall motility (66.69%), progressive motility (54.13%), viability (70.69%), membrane integrity (50.24%), acrosome integrity (80.12%) and the lowest DNA fragmentation rate (13.16%). These findings suggest that 15% chicken egg yolk in a Tris-glycerol extender optimally supports Boer goat sperm preservation. The results offer practical implications for improving artificial insemination success and establishing semen cryobanks for genetic conservation in tropical goat farming systems, particularly in regions such as the Mekong Delta, Vietnam.\u003c/p\u003e","manuscriptTitle":"Improving Boer Goat Semen Freezing Using Poultry Egg Yolks: A Study in the Mekong Delta of Vietnam","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-15 14:09:38","doi":"10.21203/rs.3.rs-7037129/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ef4e6909-0155-47bd-867c-295ded7b54b8","owner":[],"postedDate":"July 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-02T14:48:52+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-15 14:09:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7037129","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7037129","identity":"rs-7037129","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}