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10Effects of Using Commercial and Homemade Extenders on Sperm Quality of Liquid Stored Semen of Horro Chicken
Breed
Tarekegn Getachew1 * , Gebeyehu Goshu2 , and Alemayehu Lemma3
1PhD Candidate, Lecturer, Haramaya University, Haramaya, Ethiopia 2Professor of Animal Production, Addis Ababa University, College of Veterinary Medicine and Agriculture, Bishoftu, Ethiopia 3Professor of Reproductive Physiology, Addis Ababa University, College of Veterinary Medicine and Agriculture, Bishoftu, Ethiopia
Corresponding author's E-mail: [email protected]
Received: 20 November 2022 Accepted: 27 February 2023
ABSTRACT
This study aimed to evaluate the suitability of homemade tris-egg yolk-based and Commercial Beltsville poultry extenders for short-term storage of semen from the Ethiopian Indigenous Horro chicken breed at refrigeration temperature. A total of 30 Horro roosters with an average age of 40 weeks were used to collect semen. The treatments (T) in the sperm quality experiment were control (semen without extender added), semen extended with homemade extender (E1), and semen extended with commercial Beltsville Poultry Semen Extender (E2). Changes in spermatozoa motility, in vitro viability, and morphology were evaluated in fresh semen and semen diluted as 1:4 (v/v semen to extender) and stored for 4, 8, 12, and 24 hours at 4°C. During semen storage, there was a decrease in mass motility, an increase in morphologically abnormal spermatozoa with a high incidence of the bent tail, and an increase in dead spermatozoa. The commercial Beltsville poultry extender was found to be the most suitable extender regarding mass motility and in vitro viability of stored spermatozoa, but there was no significant difference in sperm abnormalities across all extenders. The results showed locally prepared tris-egg yolk-based extender could be a suitable extender for short-term storage of chicken sperm regarding the sperm quality attributes.
Keywords: Horro, In vitro viability, Motility, Morphology, Semen, Sperm
JWPR
2023, Scienceline Publication
J. World Poult. Res. 13(2): 216-222, June 25, 2023 Journal of World's
Research Paper, PII: S2322455X2300024-13 Poultry Research License: CC BY 4.0
DOI: https://dx.doi.org/10.36380/jwpr.2023.24
abdominal massage (Siudzin'ska and Lukaszewicz, 2008). Artificial Insemination in poultry reproduction has caused Growing demand for poultry products and the high rearing investigators to become interested in studying the semen
cost of breeder stock necessitates the development of modern characteristics of different poultry breeds (Haunshi, et al.,
solutions to increase production efficiency at reducing costs. 2010).
Artificial insemination is one of the solutions that significantly lowers the cost of rearing by decreasing the number of males in the flock (Lukaszewicz et al., 2020). Artificial insemination was the first great biotechnology applied to improve the reproduction of farm animals. It has an impact worldwide on many species of farm animals and endangered species (Foote, 2002). Artificial insemination technology laid the foundation for developing other reproductive technologies, such as cryopreservation and sexing of sperm, estrous cycle regulation, embryo harvesting, freezing, culture and transfer, and cloning. Artificial insemination in poultry grew significantly during the last few decades after the development of semen collection through
One of the advantages of AI application in poultry is the efficient use of males. This, in turn, decreases the cost of breeding directly by reducing the number of roosters (Benoff et al., 1981). The increasing importance of AI in poultry reproduction has caused investigators to be interested in developing the proper conditions for liquid (short-term) semen storage (Lake, 1983). The possibility of dilution and storage of poultry semen would enable poultry breeders to use superior males and inseminate many females even on distant farms (Reddy, 1995). The most common procedure for short-term storage of semen requires suspending sperm in an extended to retain their viability in vitro (Reddy, 1995). A comparison of diluted and undiluted stored semen showed
To cite this paper: Getachew T, Goshu G and Lemma A (2023). Effects of Using Commercial and Homemade Extenders on Sperm Quality of Liquid Stored Semen of Horro Chicken Breed. J. World Poult. Res., 13(2): 216-222. DOI: https://dx.doi.org/10.36380/jwpr.2023.24
that applying extenders is necessary to sustain good-quality sperm (Bilgili et al, 1987, Lukaszewicz et al., 2020). Studies have indicated that diluted poultry semen could be stored for up to 24 hours without impairing its viability and fertilizing ability (Soler et al., 2016; Silyukova et al., 2022). According to Gerzilov (2010), many factors could affect the quality of stored semen, such as the types of diluents, packaging, and cooling rates. The aim of this study was to determine the effect of two types of extenders on the qualitative characteristics of spermatozoa during short-term storage.
MATERIAL AND METHODS
Ethical approval
The present study followed institutional guidelines for humane animal treatment and complied with relevant legislation from Addis Ababa University College of Veterinary Medicine, Ethiopia.
Roosters' management
For the purpose of semen collection, thirty adult Horro roosters with an average age of 40 weeks were purchased from Debrezeit Agricultural Research Center, Bishoftu, Ethiopia. All experimental animals were managed at the poultry farm of the Debre Zeit Agricultural Research Center. The roosters were kept separately from the hens and trained for semen collection by abdominal massage technique for 2 weeks. The roosters were kept in a deep litter system and fed with a breeder ration containing 17% CP and 2800 Kcal/Kg energy (Table 1). Feed was provided twice a day with an amount of 110 gm/rooster/day, and water was provided ad libitum. All experimental chickens were dewormed and vaccinated for major diseases, including Newcastle, Marek's, Gumboro, fowl pox, and fowl typhoid (Table 2). The roosters were acclimatized for two weeks before sample collection.
Table 1. Breeder ration formula used during the experiment_
Serial number Feed ingredient_Inclusion rate (%)
1 Corn 52
2 Soy cake 10
3 Meat and bone meal 6
4 Wheat bran 15
5 Noug cake 9
6 Limestone 6
7 Breeder premix 0.5
8 Lysine 0.1
9 Methionine 0.1
10 Molasses 1
11 Salt 0.3
Breeder premix: Industrial, well-balanced premix that ensures fertile, hatching eggs and ultimately strong chicks. It contains vitamins and minerals.
Table 2. Vaccination schedule of Horro chickens in the present experiment
Age Vaccine Administration
route
Day-1 Marek Subcutaneous (neck)
Day-2 Newcastle disease Eye drop
Day-7 Gumboro Drinking water
Day-14 Newcastle (Lasota) Drinking water
Day-18 Gumboro Drinking water
Week-6 Newcastle (Lasota) Drinking water
Week-8 Fowl typhoid Injection
Week-9 Deworming Drinking water
Week-10 Fowl pox Wing stab
Week-14 Fowl typhoid Injection
The vaccines originated from the National Veterinary Institute, Bishoftu, Ethiopia
Table 3. Contents of the homemade extender
Contents Amount
Tris (base) 2.42 gm
Citric acid 1.48 gm
Fructose 4 gm
Egg-yolk 20 % v/v
Gentamicin 25 mg
Double distilled water 100 ml
pH was adjusted to 6.8
Extender preparation
The homemade extender used in this study was tris-egg-yolk-based. Semen diluents were prepared by mixing tris (base), citric acid, fructose, and egg yolk, into which an antibiotic was added. The ingredients of the extender were purchased from a local supplier. The composition of diluents is presented in Table 1. The second extender (E2) was the Beltsville commercial extender (P2-7450, continental, Delavan, WI, USA), a standard extender for the preservation of avian semen. Its composition was sodium glutamate (8.67 g/l), dipotassium phosphate (7.59 g/l), sodium acetate (3.2 g/l), fructose (5 g/l), potassium citrate (0.64 g/l), n-tris (hydroxymethyl) methyl 1-2 amino ethane sulfonic acid (TES; 3.2 g/l), monopotassium phosphate (0.7 g/l) and magnesium chloride (0.34 g/l). Osmolality and pH were set at 310 mOsmol/kg and 7.1, respectively.
Semen collection and initial evaluation
Semen was collected using the Quinn and Burrows abdominal massage technique developed in 1936. The semen was collected with a sterile tube. Two ejaculates were collected from each rooster. The ejaculate volume varies from rooster to rooster, which averages 0.3 ml. The roosters were trained for semen collection following the two weeks of acclimatization. After collection, the semen was maintained in a water bath at 37°C and subjected to on-site pre-freeze evaluation, including volume, color, pH, sperm concentration
(bill/ml), motility (%), morphological abnormality (percentage of abnormal sperms) and live percent. Qualifying ejaculates having > 60% motility, > 70% live percent, and < 30% morphological abnormality were pooled to get sufficient semen for replication and further processing (Getachew et al., 2015).
Semen processing for liquid storage assessment
After pre-freeze evaluation for semen quality attributes, qualifying semen was pooled to get 15 ml of semen aliquots. The semen aliquots were divided into three groups with 5ml each and diluted at 37°C within 10 minutes with two pre-warmed extenders (homemade extender (E1), and commercial Beltsville extender (E2) at 1:4 ratio (v/v)). A 5 ml third un-extended semen portion was set as a control. Each treatment had 5 replications. Semen was diluted immediately after initial evaluation and stored at 4°C for 4, 8, 12, and 24 hours. All the semen quality assays were performed at 4, 8, 12, and 24 hours of storage (Silyukova et al., 2022).
Semen quality assays for liquid-stored semen
Semen was first evaluated for volume (ml), color, texture, and pH. The concentration (mil/ml and billion) was measured using a hemocytometer (Counting chamber, Muhwa, China), while motility (%), viability (%), and morphology (%) were evaluated under the light microscope (MSC-P200). An eosin-nigrosin stain was used to evaluate morphology at X1000 magnification under oil immersion. A total of 200 spermatozoa were counted to determine the percentage of abnormal sperms (Siudzin'ska and Lukaszewicz, 2008).
Statistical analysis
The data collected during the study period were subjected to Analysis of Variance (ANOVA) using STATA software (version 12). Means values were compared using LSD. A significance level of 5% was used to determine statistical significance when F-test was found significant (p < 0.05). Factorial 3*4 completely randomized design was utilized to evaluate the effect of storage time and types of extenders.
Table 4. Treatments and experimental layout for fresh semen quality assessment of Horro Breed
Type of extender Time of storage 4 hours 8 hours 12 hours 24 hours
0 (Control) 0 (H4) 0 (H8) 0 (H12) 0 (H24)
E1 E1 (H4) E1 (H8) E1 (H12) E1 (H24)
E2 E2 (H4) E2 (H8) E2 (H12) E2 (H24)
E1: Extender 1; E2: Extender 2; H: Hour
RESULTS
Fresh semen characteristics
A summary of the results of semen characteristics addressed in this study is presented in Table 5. The effect of semen extenders and storage time on sperm quality is presented in Table 6.
Effect of semen extenders and storage time on sperm quality
There were significant differences in sperm motility and in vitro viability across the interactions of storage time and a group of extenders (p < 0.05). No significant difference was observed in sperm morphological abnormalities across the interactions of storage time and extenders (p < 0.05). Significantly highest sperm motility and in vitro viability rate was observed in semen extended using a commercial extender at 4 hours of storage (p < 0.05). The percentages of live sperms in treatments were observed at 83.6% and 82.6% for commercial extender extended semen and locally prepared extender extended semen, respectively. Semen extended with E2 and E1 extenders was observed consistently higher motility, compared to the control extender, irrespective of storage time.
Effect of semen extenders on sperm quality
There were significant differences (p < 0.05) in sperm motility, morphological abnormalities, and in vitro viability between the control and the two extenders. However, there was no significant difference (p < 0.05) in all semen quality parameters between the commercial and locally prepared extenders. Significantly highest sperm motility, morphological abnormalities, and in vitro viability rates were observed in semen extended samples using commercial and locally prepared extenders when compared to the control treatment (p < 0.05).
Effect of interaction of semen extenders and storage time on sperm quality
There were significant differences in progressive sperm motility across all groups of treatments (p < 0.05). There were significant differences observed in sperm morphological abnormalities between all groups except the 12 and 24 hours of storage (p < 0.05). There were also significant differences (p < 0.05) in in vitro sperm viability across the durations of storage except for between 8 and 12 hours of storage. Significantly highest sperm motility, lower morphological abnormalities, and higher in vitro viability rates were observed in semen stored for 4 hours compared to other groups (p < 0.05).
Table 5. General semen characteristics of the Horro chicken breed
Semen characteristics Mean semen characteristics
Ejaculate volume (ml) 0.36
Color Milky white
Texture Moderate viscous
Sperm total concentration/ml 5.5X109
Sperm count/ejaculate 1.98X109
Ph 7.2
Table 6. Effect of interaction of semen extenders and storage time on sperm quality of Horro chicken
Factor Mean ± SE sperm parameters Progressive motility (%) Abnormality (%) Viability (%)
Extender (storage time Significance) at p < 0.05 *** NS ***
Control (4 hours) 77 ± 2.54a 15.4 ± 1.81 73.2 ± 1.39b
Control (8 hours) 59 ± 1.87b 16.2 ± 1.80 55.8 ± 1.66c
Control (12 hours) 42 ± 1.22c 25.4 ± 2.78 49 ± 1.22c
Control (24 hours) 21 ± 1.00d 27.2 ± 1.59 11.4 ± 1.21d
E2 (4 hours) 87 ± 1.22a 10.4 ± 0.51 83.6 ± 1.63a
E2 (8 hours) 79 ± 1.00a 15 ± 1.82 77.8 ± 1.28a
E2 (12 hours) 50 ± 2.74b 17.8 ± 1.62 68.4 ± 1.50b
E2 (24 hours) 46 ± 1.87c 23 ± 2.30 51 ± 1.14c
E1 (4 hours) 84 ± 1.00a 12.2 ± 1.39 82.6 ± 1.36a
E1 (8 hours) 72 ± 1.22a 16.6 ± 1.57 73.8 ± 1.93b
E1 (12 hours) 49 ± 1.87b 19.8 ± 2.08 66.4 ± 1.50b
E1 (24 hours) 45 ± 1.58c 25.4 ± 1.21 46.4 ± 1.44c
E1: Extender 1; E2: Extender 2; NS: Non-significant; SE: standard error; abcd Different letters within the same row show significant differences among the
groups (p < 0.05).
Table 7. Effect of semen extenders on sperm quality of Horro chicken
Factor Mean ± SE sperm parameters Progressive motility (%) Abnormality (%) Viability (%)
Control 49.75 ± 4.82b 21.05 ± 1.54b 47.35 ± 5.21b
E1 65.25 ± 4.22a 16.55 ± 1.30a 70.2 ± 2.90a
E2 62.75 ± 3.71a 18.5 ± 1.32a 67.3 ± 3.15a
E1: Extender-1; E2: Extender-2; SE: Standard error; abc Different letters within the same row show significant differences among the groups (p < 0.05).
Table 8. Effect of storage time on sperm quality of Horro chickens
Factor Mean ± SE sperm parameters Progressive motility (%) Abnormality (%) Viability (%)
4 hours 82.67 ± 1.45a 12.67 ± 0.91a 79.8 ± 1.48a
8 hours 70 ± 2.34b 15.93 ± 0.95b 69.13 ± 2.70b
12 hours 47±1.44c 21 ± 1.46c 61.26 ± 2.45b
24 hours 37.33 ± 3.19d 25.2 ± 1.05c 36.26 ± 4.77c
SE: Standard error; abcd Different letters within the same row show significant differences among the groups (p < 0.05).
DISCUSSION
Semen color depend on the species of chicken used; however, generally, present findings of milky white semen were in agreement with previous reports by Peters et al. (2008) and Mussa et al. (2023). The color of domestic fowl semen varies from a dense opaque suspension to a watery fluid secreted by various reproductive glands, from a relatively high sperm density or degrees of clear to milky
white, with declining sperm numbers (Hafez and Hafez, 2000). According to Yadav et al. (2019), the color of semen may depend on the species of chicken used, but generally semen should be creamy which indicates a high sperm concentration which is in agreement with the current study. Color could also serve as an indicator of contamination (Yadav et al., 2019).
According to Peters et al. (2008), the average ejaculate volume of semen from chicken using the
abdominal massage technique was 0.01 ml to 0.35 ml in Giriraja, Frizzled feathered chicken. Bah et al. (2001) also reported an ejaculate volume of 0.28 ml in Nigerian local cocks. Cole and Cupps (1977) reported ejaculate volume within the range of 0.1 ml to 1.5 ml per ejaculation. On the other hand, Hafez and Hafez (2000) indicated that the average sperm volume collected from white leghorn varies from 0.2 to 0.5 ml. These studies are in agreement with the result found in this study on Ethipian Horro Chicken which is 0.36 ml/ejaculate.
The average sperm concentration in the present study was 5.5X109/ml. Results from Antalan et al. (2015); AL-Saeedi et al. (2019) showed that, the concentration of ranging 3.4 to 6.8X109/ml in Lohmann Brown cocks. According to Gordon (2005) reported the average sperm concentration of poultry semen was 5000X106 sperm/ml. On the other hand, the sperm concentration recorded from the present study was within the range of a report by Hafez and Hafez (2000), which is 3000-7000X106 spermatozoa/ml. The average pH of the semen collected was slightly alkaline and ranges from 7.2-7.5. Alkalinity of the poultry semen is due to the accessory sex gland fluid is generally alkaline as reported by Bah et al. (2001) and Peters et al. (2008). Results from Hafez and Hafez (2000), Gordon (2005), Antalan et al. (2015), and AL-Saeedi et al. (2019) are all within the range of the current study.
The results from the present study demonstrated the effect of a Glycerolized tris-egg-yolk-based extender on the Ethiopian indigenous Horro chicken breed's semen sperm motility, morphology, and in vitro viability. Results in this study showed that semen stored in a Glycerolized tris-egg-yolk-based extender has sperm motility that is fit for insemination. The current result of sperm motility agrees with a similar study by AL- Saeedi et al. (2019) which utilized a Tris-based extender for short-term storage of Lohmann brown breeders. As reported by Ponglowhapan et al. (2004) motility is an important indicator of sugar utilization by spermatozoa as sugars serve as an external energy source essential for maintaining motility. This study demonstrated that semen extended with a commercial extender and stored at 4 hours produced higher sperm motility (87 ± 1.22 %). In this study, the overall average sperm motility was 59.25%, which was in general agreement with 42-80% reported by Hafez and Hafez (2000).
In this study, extending semen with a commercial extender and storing it for 4 hours yielded the least sperm abnormalities (10.4 ± 0.51%). Whereas, the average sperm morphological abnormality semen stored using a
Glycerolized tris-egg-yolk-based extender was 18.5%. The number of live sperm with abnormalities in fresh cockerel semen varied from 6 to 9 percent (Tselutin et al., 1999), which was lower than the results of this study. However, Tuncer et al. (2006) reported that the number of abnormal sperm in cockerel semen varied from 9.2 to 11.23%, which is in agreement with sperm abnormalities recorded in semen extended using a commercial extender.
A commercial extender at 4 hours of storage was the best combination (83.6 ± 1.63%) for better in vitro sperm viability as compared to other treatments. Sperm in vitro viability recorded using LPE at 4 hours of storage was slightly lower than that of commercial extenders (82.6 ± 1.36%). The LPE improved the longevity of sperm in this study as Bearden et al. (2004) reported "presence of fructose will not greatly change the metabolic rate, however, will extend the life span of the sperm". According to the report by Gebriel et al. (2009), 81.79% of sperm in vitro viability was recorded at 6 hours of storage, which was a similar to results of the present study. In this study, the percentage of dead sperm increased by 36.2% over 24 hours of storage for semen extended with LPE and which was positively correlated with the storage time. In general, the results of sperm quality attributes observed in this study are comparable to several studies (Lukaszewicz et al., 2008; AL- Saeedi et al., 2019).
CONCLUSION
In this study, tris-egg-yolk-based LPE yielded comparable results in all sperm quality attributes when compared with commercial Beltsville Poultry Semen extender. Semen stored for more than 12 hours at refrigeration temperature showed significantly lower sperm quality. Semen stored using a commercial extender for 4 hours was recorded with a higher level of sperm quality. Further studies are recommended to explore the possible ways to store poultry semen for 24 hours at refrigeration temperature without decreased sperm quality significantly.
DECLARATION
Acknowledgments
The authors are grateful to Addis Ababa University and Debrezeit Agricultural Research Institute, Bishoftu, Ethiopia for arranging experimental area. This study was funded by Ministry of Education, Ethiopia.
Authors' contributions
Tarekegn Getachew, Gebeyehu Goshu and Alemayehu Lemma designed the experiments and
Tarekegn Getachew performed the experiments. Tarekegn Getachew derived the models and analyzed the data. Gebeyehu Goshu and Alemayehu Lemma assisted with standardizing data collection and data analysis. Tarekegn Getachew wrote the manuscript in consultation with Gebeyehu Goshu and Alemayehu Lemma. All authors read and approved the final version of the manuscript for publishing in the present journal.
Competing interests
The authors have declared that no competing interest exists.
Ethical consideration
All ethical issues, including plagiarism, consent to publish, misconduct, data fabrication and/or falsification, double publication and/or submission, and redundancy, have been checked by all authors.
Availability of data and materials
The data that support the findings of this study are available from the corresponding author, T. Getachew, upon reasonable request.
REFERENCES
AL- Saeedi TA, AL-Juaifari AAI, and Al-mahmoudi AHJ (2019). The effect of different extenders on some fertility properties of rooster semen. International Journal of Poultry Science, 18(11): 504-507. DOI: https://www.doi.org/10.3923/ijps.2019.504.507
Antalan DG, Aquino FP, Leoveras EDC, Ocampo LC, Atabay EP, and De Leon AM (2015). The effect of semen extender and storage time on the quality of spermatozoa collected from the excurrent duct of Philippine local chicken. Journal of Biological Engineering Research and Review, 2(2): 18-20. Available at:
https://wwwbiologicalengineering.in/gallery/15-2-2-05.pdf
Bah GS, Chaughari SUR, and Al-Amin JD (2001). Semen characteristics of local breeder cocks in the Sahel region of Nigeria. Revue d'élevage et de médecine vétérinaire des pays tropicaux, 54(2): 153-158. DOI: https://www.doi.org/10.19182/remvt. 9794
Bearden HJ, Fuquay JW, and Willard ST (2004). Applied animal reproduction, 6th Edition. Pearson Education, Inc., New Jersey, pp. 109-127. Available at: https://www. worldcat.org/title/applied-animal-reproduction/oclc/469432992
Benoff FH, Rowe K, Fuguay JI, Renden JA, and Scott AR (1981). Effect of semen collector on semen volume and sperm concentration in broiler breeder males. Poultry Science, 60(5): 1062-1065. DOI:
https://www.doi.org/10.3382/ps.0601062
Bilgili, SF, Sexton KJ, and Renden JA (1987). Fluorometry of poultry semen: Influence of dilution and storage on chicken spermatozoa viability and fertility. Poultry
Science, 66(12): 2032-2035. DOI:
https://www.doi.org/10.3382/ps.0662032
Cole HH and Cupps PT (1977). Reproduction in domestic animals, 4th Edition. Academic press., New York, p. 195. Available at:
https://www.elsevier.com/books/reproduction-in-domestic-animals/cupps/978-0-08-057109-6
Foote RH (2002). The history of artificial insemination: Selected notes and notables. American Society of Animal Science, Cornell University, Ithaca, NY, USA, pp. 1-10.
https ://www.asas. org/docs/default-
source/midwest/mw2020/publications/footehist.pdf?sfvrsn=59da6c07 0
Gebriel GM, Kalamah MA, El-Fiky AA, and Ali AFA (2009). Some factors affecting semen quality traits in Norfa cocks. Egyptian Poultry Science Journal, 29(2): 677-693. Available at:
https://www.cabdirect.org/cabdirect/abstract/20113110365
Gerzilov P (2010). Influence of various cryoprotectants on the sperm mobility of Muscovy semen before and after cryopreservation. Agricultural Science and Technology, 2(2): 57-60. Available at:
https://www.cabdirect.org/cabdirect/abstract/20113272102
Getachew T, Ameha N, and Tefera M (2015). Fructose addition in skim milk based extender and its effect on semen quality in White Leg-Horn chicken. Ethiopian Veterinary Journal, 19(1): 23-34. DOI:
https://wwwdoi.org/10.4314/evj.v19iL2
Gordon I (2005). Reproductive technologies in farm animals. CABI Publishing., UK, Chapter 1, pp. 16-28. Available at: https://www.cabi.org/animalscience/ebook/20173185376
Hafez B and Hafez ESE (2000). Reproduction in farm animals, 7th Edition. Lippincott Williams and Wilkins., New York, USA, pp. 267-292. Available at: https://b2n.ir/g36564
Haunshi S, Doley S, Kadirvel G (2010). Comparative studies on egg, meat, and semen qualities of native and improved chicken varieties developed for backyard poultry production. Tropical Animal Health and Production, 42: 1013-1019. Available at:
https://link.springer.com/article/10.1007/s11250-009-9524-3
Lake PE (1983). Factors affecting the fertility level in poultry, with special reference to artificial insemination. Agricultural Research Council, 39(2): 106-117. DOI: https://www.doi.org/10.1079/WPS19830011
Lukaszewicz E, Jersey A, Partyka A, and Siudzin'ska A (2008). Efficacy of evaluation of rooster sperm morphology using different staining methods. Research in Veterinary Science, 85(3): 583-588. DOI:
https://www.doi.org/10.1016/j.rvsc.2008.03.010
Lukaszewicz E, Jerysz A, and Kowalczyk A (2020). Effect of semen extenders on viability of ISA Brown and Hubbard Flex roosters' sperm stored for 24 h. Poultry Science, 99(5): 2766-2774. DIO:
https://www.doi.org/10.1016/j.psj.2019.12.055
Mussa NJ, Boonkum W, and Chankitisakul V (2023). Semen quality traits of two Thai native chickens producing a high and a low of semen volumes. Veterinary Sciences, 73(10): 1-9. DOI: https://www.doi.org/10.3390/vetsci10020073
Peters SO, Shoyebo OD, Ilori BM, zoje MOO, Ikeobi CON, and Adebambo OA (2008). Semen quality traits of seven strain
of chickens raised in humid tropics. International Journal of Poultry Science, 7(10): 949-953. DOI: https://www.doi.org/10.3923/ijps.2008.949.953
Ponglowhapan S, Birgitta E, and Catharina LF (2004). Influence of glucose and fructose in the extender during long-term storage of chilled canine semen. Theriogenology, 62(8): 1498-1517. DOI:
https://www.doi.org/10.1016/j.theriogenology.2004.02.014
Quinn JP and Burrows WH (1936). Artificial insemination in fowls. Journal of Hered, 27(1): 31-38.DOI: https://www.doi.org/10.1093/oxfordiournals.ihered.a104138
Reddy RP (1995). Artificial insemination of broilers: Economic and management implications. In: M. R. Bakst (Editor), Poultry Science Association, pp. 73-89.
Silyukova M, Galimov R, Kuzmina T, Kulikova K, Nizhnikov A, and Chernyshova O (2022). Proteomic characterization of chicken spermatozoa reveals potential biomarkers of semen quality. Reproduction, Fertility and Development, 34(1): 153-165. DOI: https://www.doi.org/10.1071/RD20300
Siudzin'ska A and Lukaszewicz E (2008). Effect of semen extenders and storage time on sperm morphology of four chicken breeds. Journal of Applied Poultry Research,
17(1): 101-108.
https://www.doi.org/10.3382/iapr.2007-00048
DOI:
Soler C, García-Molina A, Valverde A, Núñez-Martínez I, Fernández-Santos MR, Esteso MC, ... and Pérez-Gutiérrez JF (2016). Seminal plasma proteins as markers of sperm fertility. Theriogenology, 85(8): 1466-1473. DOI: https://www.doi.org/10.1016/j.theriogenology.2016.01.025
Tselutin K, Seigneurin F, and Blesbois E (1999). Comparison of cryoprotectants and methods of cryopreservation of fowl spermatozoa. Poultry Science, 78(4): 586-590. DOI: https://www.doi.org/10.1093/ps/78A586
Tuncer PB, Kinet H, Ozdogan N, and Demiral O (2006). Evaluation of some spermatological characteristics in Denizli cocks. Journal of The Faculty of Veterinary Medicine Erciyes University, 3(1): 37-42. Available at: https://citeseerx.ist.psu.edu/document?repid=rep1&type=p df&doi=b597d4efb95049f0d30d199ae9d2d8e774945eb0
Yadav S, Kumar R, Gupta AK, and Yadav SK (2019). Advances in poultry semen preservation: conventional and emerging technologies. Journal of Animal Science and Technology, 61(6): 331-340. https://doi. org/10.5187/j ast.2019.61.6.331
