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. 2023 Nov 7;103(1):103262. doi: 10.1016/j.psj.2023.103262

Improving the quality of chilled semen from Thai native chicken using phosphorus and vitamin B12 supplementation in semen extender

Junpen Suwimonteerabutr ⁎, Unchean Yamsrikaew †, Khemiga Damthongsen †, Thornjutha Suksirisamphan †, Paniga Leeniwa †, Pawita Lawanyakul †, Morakot Nuntapaitoon ⁎,1
PMCID: PMC10801650  PMID: 38007902

Abstract

This study aimed to determine phosphorus and vitamin B12 supplementation effect in semen extender on the quality and fertility ability of chilled Thai native rooster semen. Eighty-four ejaculates of semen from 26 Thai native roosters (Burmese × Vietnam crossbreed) were included. Semen was collected by applying dorsal–abdominal massage once a week, pooled, diluted to 500 million sperms per dose, and divided into 6 groups. The semen samples used for control group were diluted with modified Beltsville poultry semen extender (BPSE). For the treatment groups 2 to 6: semen samples were diluted with modified BPSE and enriched with phosphorus and vitamin B12 (Octafos Octa Memorial Co., Ltd., Bangkok, Thailand) at concentrations 0.02, 0.04, 0.06, 0.08, and 0.10%. Semen fertility ability was tested in 6 replications by inseminating layer hens. Thirty-six Thai native hens were randomly assigned to 3 groups (control, 0.04, and 0.08%) of 12 hens and were inseminated with a dose of 0.2 mL on collecting day. Sperm motion characteristics (i.e., sperm motility, sperm progressive motility, and sperm kinetic parameters) were measured using a computer-assisted sperm analysis system (SCA, Proiser S.L., Valencia, Spain). Sperm viability, mitochondrial activity, acrosome integrity, plasma membrane integrity, and malondialdehyde (MDA) concentration were also evaluated. The sperm motion characteristics were the highest in the 0.04% supplementation group on all days of collection, especially the VCL and VAP (P < 0.05). The viability, mitochondrial activity, plasma membrane and acrosome integrity of spermatozoa were greater in the 0.04% supplementation group than in the control groups (P < 0.05). The 0.04% supplementation group had the lowest MDA concentration in all days of collection. The 0.04% supplementation group were higher both fertility (66.59 vs. 48.50%: P < 0.05) and hatching rates (58.80 vs. 43.18%: P < 0.05) than in the control group. In conclusion, 0.04% phosphorus and vitamin B12 concentrations supplementation in semen extender improved rooster semen quality and fertility in chilled rooster semen.

Key words: chilled semen, extender, phosphorus, Thai native rooster, vitamin B12

INTRODUCTION

Artificial insemination (AI) is a valuable tool in many species (i.e., swine, cattle, equine, and avian) for preventing the spread of contagious diseases, improving genetics, and increasing animal production. AI in roosters is usually applied for preserving semen from animals threatened with extinction (Parkinson and Morrell, 2019) and for increasing breeding efficiency and productivity in native poultry. Thai native chicken have a long history of breeding and cultural significance; their ability to thrive in hot and humid climates make them popular in tropical regions. Furthermore, Thai native chicken breeds have gained global interest due to their unique characteristics. Semen preservation is currently used as a tool to preserve the genetic information of chicken worldwide. However, chilled and cryopreservation techniques are still under development. Storing rooster semen in a liquid form at 4°C aims to retard sperm metabolism maintain its viability for a longer period compared with storing at room temperature. However, sperm viability and function still decline during the 48 h storage at 4°C after semen collection (Blesbois and Brillard, 2007). Many chemicals were added in the extender for increasing chilled semen quality in rooster, for example, coenzyme Q10, oleic acid and phosphatidylcholine (Long and Conn, 2012; Eslami et al., 2016; Masoudi et al., 2019; Sharideh et al., 2019). Coenzyme Q10 is an important role in protective effect against free radicals and energy production as oleic acid (Mancini et al., 2005). Furthermore, the beneficial effects of oleic acid are related to the antioxidant activity and signal transduction alteration (Ruiz-Gutiérrez et al., 1999). Meanwhile, phosphatidylcholine in rooster semen as membrane-bound phospholipids is lost during storage (Blesbois et al., 1999).

The sperm membrane of rooster is composed of a high concentration of poly-unsaturated fatty acids (PUFAs) which easily to be undergo lipid peroxidation (LPO) during storage, resulting in decreased sperm motility because of reduced ATP production (Masoudi et al., 2019). Phosphorus provides energy for metabolism to sperm cells by being part of high-energy compounds (adenosine triphosphate, ATP; adenosine monophosphate, AMP), creatine phosphate, and nucleotides (Krämer, 1996; Klingenberg, 2008). It is also a component of glycerylphosphorylcholine (GPC), which is found in semen, synthesized by the epithelial cells of epididymis, and is an indicator of semen function (Getachew, 2016). GPC level is positively correlated with sperm motility (Arrata et al., 1978). Numerous studies found that phosphorus supplementation enhances semen efficiency in many species (López Rodríguez et al., 2013; Cazales et al., 2015; Beltrame et al., 2019; Suwimonteerabutr et al., 2020). The GPC was applied in frozen semen for enhancing sperm survival and reducing the harmful effects of lipid peroxidation during semen storage (Long and Conn, 2012).

Vitamin B12 or cyanocobalamin is an antioxidant essential for genetic materials in spermatogenesis (Hu et al., 2011; Hamedani et al., 2013). It is a water-soluble vitamin functioning as a coenzyme in a number of biochemical reactions (Hu et al., 2009). Vitamin B12 is also used in gluconeogenesis and Krebs cycle (Kennedy et al., 1990; Mcdowell, 2000), it impairs reactive oxygen species (ROS), and is positively related with sperm quality, concentration, and fertility rates in humans (Chen et al., 2001; Boxmeer et al., 2009). Watanabe et al. (2003) reported that vitamin B12-deficient mice have a high count of abnormal shaped spermatozoa and low sperm motility and velocity. A combination of phosphorus and vitamin B12 was applied to improve semen qualities and successfully enhance sperm total motility, progressive motility, and sperm kinetics in chilled boar semen (Suwimonteerabutr et al., 2020). Phosphorus and vitamin B12 supplementation enhanced sperm motility (Cazales et al., 2015) in horses. However, the effects of phosphorus and vitamin B12 supplementation in native rooster semen have not been studied. Therefore, this study aimed to determine the effect of the combination of phosphorus and vitamin B12 supplementation in rooster semen extender on the sperm quality and fertility of chilled semen from Thai native rooster.

MATERIALS AND METHODS

Ethics Statement

The use of animals for this experiment was approved by the Institutional Animal Care and Use Committee at the Faculty of Veterinary Science, Chulalongkorn University (Approval number 2131018) following the guidelines documented in “The Ethical Principles and Guidelines for the Use of Animals for Scientific Purposes” edited by the National Research Council of Thailand.

Animals

This study was performed in a local Thai native rooster farm located in the western part of Thailand. Eighty-four ejaculates of semen from 26 Thai native roosters (Burmese × Vietnam crossbreed) aged between 1 and 3 yr were included in the experiment conducted between February 2022 and May 2022. Thai native roosters were kept in individual cages, fed 130 g commercial feed daily, and received water ad libitum. Meanwhile, 36 individually caged Thai native hens (1- and 3-yr old) with egg production rate of 90% were fed 110 g of commercial feed daily and received water ad libitum.

Semen Collection and Experimental Design

Semen was collected in a 1.5 mL microtube by applying dorso-abdominal massage once a week and was evaluated for concentration and motility. The semen samples with >65% of total motility without feces or blood and ≥85% normal morphology was used in this study. All the collected semen samples were pooled and diluted to 500 million spermatozoa per dose and divided into 6 groups. For the control group, semen was diluted with modified Beltsville poultry semen extender (BPSE). For the treatment groups 2 to 6: semen samples were diluted with modified BPSE added with 1 mL of phosphorus and vitamin B12 supplementation containing 100 mg of phosphorus, 0.05 mg of vitamin B12, and 1 mg of methyl paraben (Octafos Octa Memorial Co., Ltd., Bangkok, Thailand) at concentrations of 0.02, 0.04, 0.06, 0.08, and 0.10%. All semen samples were stored at 4°C until analysis.

Sperm Evaluation

The samples were evaluated for sperm motion characteristics and sperm characteristics on d 0, 1, 2, and 3 after collection.

Sperm Motion Characteristics

Sperm motion characteristics (i.e., sperm motility, sperm progressive motility, and sperm kinetic parameters) were measured using the CASA system (SCA, Proiser S.L., Valencia, Spain). Sperm kinetic parameters included the curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), amplitude of lateral head (ALH), linearity (LIN), straightness (STR), wobble (WOB), beat cross frequencies (BCF), and hyperactivity (HPA) (Amini et al., 2015).

Sperm Characteristics

Sperm Viability

Sperm viability was assessed as live sperm percentage using SYBR-14/propidium iodine (PI) (Chalah et al., 1998; Santiago-Moreno et al., 2018). In brief, 4 μL of SYBR-14 (0.02 mM) and 2 μL of PI (2.4 mM) were added in 100 μL of HEPES-buffered medium (130 mM NaCl, 4 mM KCl, 14 mM Fructose, 10 mM HEPES, 1 mM CaCl2, 0.5 mM MgCl2, 0.1% BSA). Meanwhile, 10 μL of rooster semen was diluted with 200 μL of phosphate buffered saline (PBS), and 10 μL of this diluted semen was mixed with 20 μL of SYBR-14/PI in HEPES-buffered medium. The remaining diluted semen was stored at 37°C for 15 min and recorded by counting a total of 200 sperm under fluorescence microscope with 400× magnification. Live spermatozoa with intact plasma membrane were stained green by SYBR-14, and those with damaged plasma membrane were stained red–green by SYBR-14 and PI. The dead spermatozoa with damaged plasma membrane were stained red by PI.

Mitochondrial Activity

JC-1 dye color (Molecular Probes, Molecular Probes Inc., Eugene, OR) was used by preparing 0.153 mmol JC-1 in DMSO, 0.02 mmol SYBR-14, and 2.4 mmol PI. The mixture of 1.6 µL of JC-1 in DMSO, 1 µL of SYBR-14, and 1.6 µL of PI 1.6 μL was diluted with 100 µL of HEPES-buffered medium. Afterward, 12.5 μL of semen and 25 μL prepared stains were combined and stored at 37°C for 30 min. Mitochondrial function of 200 spermatozoa was examined at fluorescence microscope's 400× magnification. Spermatozoa with midpiece and low mitochondrial function were stained green, and those with midpiece and high mitochondrial function were stained orange (Huo et al., 2002).

Acrosome Integrity

Acrosome integrity was assessed as the percentage of sperm cells with intact acrosome identified by Coomassie blue (Merck, Germany) staining (Abouelezz et al., 2015) with some modifications. In brief, 100 mL of staining solution was prepared by mixing 22.5 mL of 0.5% Coomassie blue, 22.5 mL of methanol, and 54.75 mL of distilled water, followed by the addition of 0.5 mL of glacial acetic acid. A drop of 10 μL of diluted semen sample was smeared on a glass slide and allowed to dry. The smear was fixed at room temperature in buffered 4% glutaraldehyde in PBS for 30 min and air dried. Afterward, the slide was stained with Coomassie blue solution mixed with 0.25% acetic acid for 2 min, rinsed with distilled water, and air dried. Thereafter, counts of 200 spermatozoa with intact acrosome under light microscope at 1,000× magnification with oil immersion were recorded. Spermatozoa with a hooked, swollen, thinned, or no acrosome were classified as lacking acrosome integrity.

Plasma Membrane Integrity

Plasma membrane integrity was assessed using 100 mOsm/kg hypoosmotic solution prepared by mixing 1 g of sodium citrate and 100 mL of double-distilled water. A drop of 3 µL of diluted semen was mixed with 100 µL of 100 mOsm/kg. The hypoosmotic solution was incubated at 37°C for 30 min. A drop of the incubated solution was then smeared on a slide and allowed to dry. The slide was stained with Coomassie blue mixed with 0.25% acetic acid for 2 min, and the results were recorded by counting 200 spermatozoa under a light microscope at 1,000× magnification. Spermatozoa classified as coiled midpieces and tail segments were positive, and those without coiled tail were negative (Sharideh et al., 2019).

Malondialdehyde Measurement

The malondialdehyde (MDA) concentration was measured using the thiobarbituric acid (TBA) reaction, using the method described by Kheawkanha et al. (2023). Briefly, the spermatozoa suspension (250 × 106 spz/mL) was mixed with 0.25 mL of 0.2 mm ferrous sulfate and 0.25 mL of 1 mm ascorbic acid, then incubated in water bath at 37°C for 60 min. After incubation, the tubes were consecutively added with 1 mL of 15% (w/v) trichloroacetic acid (TCA) and 1 mL of 0.375% (w/v) thiobarbituric acid (TBA), 0.25 N HCl. The mixture was shaken. After that, tubes were heated in boiling water for 10 min then cooled down in ice bath for 5 min to stop the reaction. Finally, the samples were centrifuged at 800 × g for 10 min. The absorbance of the upper layer was read at 532 nm wavelength. The amounts of MDA were expressed as nmol/mL by 250 × 106 spz/nmol/mL in the samples.

Fertility

The fertility ability of chilled semen was tested by inseminating Thai native hens. Thirty-six hens were randomly assigned to 3 groups (control, 0.04%, and 0.08%) of 12 hens and inseminated with a dose of 0.2 mL on collecting day. All inseminations with 0.10 mL (200 × 106 spermatozoa/dose) were performed at 10:00 am and 4:00 pm. Eggs were collected 2 to 10 d after insemination. Fertilization was determined by candling eggs on d 7 of incubation. Six replications of fertility test were carried out. The hatching rate was determined based on fertilized eggs on d 21 of incubation.

Statistical Analysis

Statistical analyses were carried out using SAS (SAS version 9.1, Cary, NC). The effect of phosphorus and vitamin B12 on sperm motion characteristics, sperm characteristics and MDA on each day after collection were analyzed by the general linear mixed model (MIXED). Statistical modeling included experimental groups (control, 0.02, 0.04, 0.06, 0.08, and 0.10% of phosphorus and vitamin B12); day of collection (0, 1, 2, and 3); and interaction between experimental groups and day of collection. Thai native rooster was included as a random variable. Additionally, the effect of phosphorus and vitamin B12 on fertility and hatch ability were analyzed using multiple ANOVA. Least square means were obtained from each class of the factor and compared using the least significant test. Differences with values of P < 0.05 were considered statistically significant.

RESULTS

Descriptive Statistics

The average semen volume in Thai native roosters was 0.2 ± 0.1 mL (range 0.01–0.50 mL), the motility was 71.5 ± 5.8% (range 65.0–85.0%), and the concentration was 1,954.8 ± 948.0 × 106 sperms/mL (range 20–3,980 × 106 sperms/mL). The percentages of total motility and progressive motility analyzed by CASA were 66.6 ± 8.0% and 14.7 ± 4.7%, respectively. The sperm motility and sperm kinetic parameters analyzed by CASA in the 0.04% supplementation group were significantly higher than those in the control group (Table 1). All sperm parameters decreased on the day of collection (P < 0.001).

Table 1.

Effect of phosphorus and vitamin B12 supplementation concentration in semen extender and storage duration on sperm motility and sperm kinetic parameters analyzed by CASA.

Parameters Groups
 SEM1 Day of storage
 SEM1
Con 0.02 0.04 0.06 0.08 0.10 0 1 2 3
Total motility, % 60.3d 63.9bc 66.4a 64.5b 65.0b 63.2c 2.7 69.4a 65.1b 61.9c 59.0d 2.7
PR⁎⁎, % 12.1d 14.4c 16.1a 15.2b 14.7bc 14.1c 1.3 17.8a 14.8b 13.2c 11.9d 1.3
VCL, µm/s 53.0d 55.2bc 57.2a 56.2b 55.3bc 54.9c 1.3 58.1a 55.8b 54.4c 52.9d 1.3
VSL, µm/s 14.3c 15.2b 15.8a 15.7a 15.3b 15.2b 0.4 16.5a 15.3b 14.9c 14.3d 0.4
VAP, µm/s 24.5d 25.9c 27.1a 26.6ab 26.0bc 25.8c 0.7 27.8a 26.1b 25.4c 24.6d 0.7
LIN, % 28.0b 28.5ab 28.8a 29.0a 28.7a 28.7ab 0.3 28.9 28.4 28.6 28.6 0.3
STR, % 53.0b 53.5ab 53.8a 54.1a 53.8a 53.5ab 0.3 54.1a 53.5b 53.6ab 53.3b 0.3
WOB, % 46.8b 47.4a 47.9a 47.9a 47.5a 47.4a 0.3 48.0a 47.2b 47.4b 47.3b 0.3
ALH, µm 3.47d 3.59c 3.71a 3.66ab 3.60bc 3.59c 0.07 3.73a 3.62b 3.58b 3.48c 0.06
BCF, beats/s 5.00d 5.24ab 5.32a 5.20b 5.16bc 5.09cd 0.14 5.51a 5.19b 5.02c 4.96c 0.13
HPA, % 1.32c 1.66b 1.95a 1.75b 1.74b 1.65b 0.19 2.05a 1.75b 1.57c 1.35d 0.19
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a–d

Different superscript letters within rows indicate significant difference (P < 0.05).

1

Maximum standard error of the mean (SEM).

Effect of Different Concentrations of Phosphorus and Vitamin B12 Supplementation and Day of Storage on the Sperm Motility of Chilled Semen Rooster

The percentages of total and progressive motility in the 0.04% supplementation group were the highest in all days of collection (Figure 1). On d 0 after collection, the percentages of total motility and progressive motility were significantly higher in all supplementation groups than in the control group (P < 0.05), except for the total motility of 0.10% supplementation group (Figure 1A). Differences in progressive motility were found between control and all supplementation groups on all days of the experiment (P < 0.05), with the exception on d 1 post collection (Figure 1B).

Figure 1.

Figure 1

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Effect of different concentrations of phosphorus and vitamin B12 supplementation (0 (control), 0.02, 0.04, 0.06, 0.08, and 0.10%) on sperm motility (A) and progressive motility (B) in chilled semen rooster on specific days after collection. a–cDifferent superscript letters within day indicate significant differences (P < 0.05).

Effect of Different Phosphorus and Vitamin B12 Supplementation Concentrations and Day of Storage on the Sperm Kinetic Parameters of Chilled Rooster Semen

The effect of phosphorus and vitamin B12 supplementation in chilled rooster semen on sperm kinetic parameters was assessed by CASA system on specific days after collection, and the results are presented in Tables 2 and 3. All concentrations of supplementation in semen extenders, especially 0.04% supplementation, had greater sperm kinetic parameters than the control group in all days after collection. On d 0 post collection, some sperm kinetic parameters (Table 2) such as VCL, VSL, VAP, ALH, BCF, and HPA were significantly higher than detected in the control group (P < 0.05). VCL and VAP values in the 0.04% supplementation group were higher than those in the control group in all day post collection (P < 0.05).

Table 2.

Effect of phosphorus and vitamin B12 supplementation concentration in semen extender and day of storage on the velocity of spermatozoa at d 0 and 1.

Parameters D 0
 SEM1 D 1
 SEM1
Con 0.02 0.04 0.06 0.08 0.10 Con 0.02 0.04 0.06 0.08 0.10
VCL, µm/s 54.8c 59.2a 60.4a 59.0a 59.0ab 57.9b 1.4 55.0b 55.4ab 57.1a 56.4ab 55.5ab 55.2ab 1.4
VSL, µm/s 15.3b 16.8a 17.0a 17.1a 16.3a 16.4a 0.5 14.8 14.9 15.6 15.5 15.5 15.6 0.5
VAP, µm/s 25.9d 28.3abc 29.1a 28.6ab 27.5bc 27.4c 0.8 25.4b 25.6b 26.8a 26.5ab 26.3ab 26.0ab 0.8
LIN, % 28.4 29.1 28.9 29.5 28.7 29.0 0.5 28.2 27.8 28.2 28.4 29.1 28.7 0.5
STR, % 53.5b 54.3ab 54.0ab 54.5a 53.8ab 54.0ab 0.5 53.6 53.0 53.3 53.4 54.2 53.8 0.5
WOB, % 47.4b 48.1ab 48.4ab 48.6a 47.6ab 47.9ab 0.5 47.0ab 46.6b 47.7ab 47.5ab 47.9a 47.1ab 0.5
ALH, µm 3.54c 3.78ab 3.88a 3.78ab 3.70b 3.68b 0.08 3.56 3.59 3.70 3.69 3.62 3.59 0.08
BCF, beats/s 5.26b 5.59ab 5.71a 5.69a 5.37b 5.42b 0.16 5.16 5.32 5.25 5.14 5.15 5.13 0.16
HPA, % 1.55d 2.25ab 2.54a 1.92cd 2.15bc 1.88bd 0.23 1.59 1.71 1.82 1.88 1.70 1.82 0.23
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a–d

Different superscript letters within rows indicate significant difference (P < 0.05).

1

Maximum standard error of the mean (SEM).

Table 3.

Effect of phosphorus and vitamin B12 supplementation in semen extender and day of storage on the velocity of spermatozoa at d 2 and 3.

Parameters D 2
 SEM1 D 3
 SEM1
Con 0.02 0.04 0.06 0.08 0.10 Con 0.02 0.04 0.06 0.08 0.10
VCL, µm/s 51.8c 54.6b 56.7a 55.1ab 54.9ab 53.8b 1.4 50.3c 51.7bc 54.7a 54.1a 52.9ab 53.6ab 1.4
VSL, µm/s 13.4b 14.9a 15.5a 15.5a 15.1a 14.8a 0.5 13.6c 14.0bc 15.2a 14.5ab 14.4abc 14.0bc 0.5
VAP, µm/s 23.4b 25.7a 26.5a 26.0a 25.6a 25.4a 0.8 23.4c 24.0bc 25.8a 25.1ab 24.7ab 24.5bc 0.8
LIN, % 27.2b 28.7a 28.9a 29.5a 28.6a 28.9a 0.5 28.3 28.4 29.3 28.7 28.6 28.9 0.5
STR, % 52.0b 53.7a 54.0a 54.6a 53.8a 53.4a 0.5 53.0 53.0 53.9 53.5 53.4 53.8 0.5
WOB, % 45.8b 47.8a 47.7a 48.0a 47.1a 48.0a 0.5 47.0ab 47.1ab 48.1a 47.5ab 47.3ab 46.7b 0.5
ALH, µm 3.42b 3.61a 3.69a 3.61a 3.58a 3.57a 0.08 3.35b 3.38b 3.59a 3.56a 3.49ab 3.53a 0.08
BCF, beats/s 4.81b 5.03ab 5.17a 5.03ab 5.11a 4.95ab 0.16 4.76c 5.01ab 5.17a 4.96ac 5.00ab 4.89bc 0.16
HPA, % 1.18b 1.49b 1.99a 1.59a 1.68a 1.47ab 0.23 0.98b 1.19b 1.47ab 1.60a 1.45ab 1.42ab 0.23
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a–c

Different superscript letters within rows indicate significant difference (P < 0.05).

1

Maximum standard error of the mean (SEM).

Effect of Different Concentrations of Phosphorus and Vitamin B12 Supplementation and Day of Storage on Sperm Characteristics

Phosphorus and vitamin B12 supplementation in semen extenders significantly affected the characteristics of spermatozoa (Table 4). The results showed that the spermatozoa's viability, mitochondrial activity, plasma membrane, and acrosome integrity were greater in the 0.04% supplementation group than in the control and other supplementation groups (P < 0.05). On d 0 post collection, viability and mitochondrial activity were higher in all the supplementation groups than in the control group (P < 0.05) (Figure 2A and B). The 0.04% supplementation group had the highest viability, mitochondrial activity, and plasma membrane (Figure 2A–C). The 0.10% supplementation group had the lowest plasma membrane integrity among the supplementation groups (Figure 2C). Phosphorus and vitamin B12 supplementation had no effect on acrosome integrity on d 0 to 2 (Figure 2D).

Table 4.

Effect of phosphorus and vitamin B12 supplementation concentration in semen extender and day of storage on the characteristics of spermatozoa and malondialdehyde (MDA) concentration.

Parameters Group
 SEM1 Day
 SEM1
Con 0.02 0.04 0.06 0.08 0.10 0 1 2 3
Viability, % 65.5e 69.2c 71.6a 70.0b 69.7bc 67.9d 1.6 73.3a 70.1b 67.9c 64.7d 1.6
Mitochondria, % 68.0d 71.7c 74.2a 72.5b 73.1b 71.4c 1.6 75.8a 72.8b 71.0c 67.7d 1.6
Membrane, % 57.6d 62.2b 64.7a 62.7b 62.3b 59.3c 1.4 66.0a 62.9b 59.9c 57.1d 1.4
Acrosome, % 95.2c 96.2b 96.7a 96.6ab 96.4ab 95.3c 0.6 97.4a 96.8b 95.8c 94.1d 0.6
MDA, nmol/mL 2.98c 2.85c 2.68c 3.23ab 2.94bc 3.50a 0.30 2.37c 2.81b 3.05b 3.58a 0.32
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a–d

Different superscript letters within rows indicate significant difference (P < 0.05).

1

Maximum standard error of the mean (SEM).

Figure 2.

Figure 2

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Effect of different concentrations of phosphorus and vitamin B12 supplementation (0 (control), 0.02, 0.04, 0.06, 0.08, and 0.10%) on sperm viability (A) mitochondrial activity (B) plasma membrane integrity (C) and acrosome integrity (D) in chilled semen rooster on specific days after collection. a–dDifferent superscript letters within day indicate significant differences (P < 0.05).

Effect of Different Concentrations of Phosphorus and Vitamin B12 Supplementation and MDA

Phosphorus and vitamin B12 supplementation in semen extenders significantly affected MDA (Table 4). The results showed that the MDA concentration was lower in the 0.04% supplementation group than in the 0.06 and 0.10% supplementation group (P < 0.05). The MDA concentration was less (P < 0.05) on d 0 post collection than other days. Additionally, the MDA concentration was greater at d 3 compared to the other days post collection (P < 0.001). The interactions between interaction between experimental groups and days of collection indicated the MDA concentration did not differ among the treatment groups at all days of collection (Figure 3, P > 0.05). On d 0 post collection, the MDA concentration was highest in 0.10% supplementation groups than in the control group (P > 0.05) (Figure 3). The 0.04% supplementation group had the lowest MDA concentration whereas, the 0.10% supplementation group had the highest MDA concentration among the supplementation in all days of collection.

Figure 3.

Figure 3

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Effect of different concentrations of phosphorus and vitamin B12 supplementation (0 (control), 0.02, 0.04, 0.06, 0.08, and 0.10%) on malondialdehyde concentration (MDA) concentration in chilled semen rooster on specific days after collection. a–cDifferent superscript letters within day indicate significant differences (P < 0.05).

Effect of Different Concentrations of Phosphorus and Vitamin B12 Supplementation on Fertility and Hatching Ability

The fertility and hatching rates of chilled semen in the 0.04% supplementation group was higher than those of the control and chilled semen with 0.08% supplementation of phosphorus and vitamin B12 (P < 0.05) (Table 5).

Table 5.

The effect of phosphorus and vitamin B12 supplementation concentration in semen extender on fertility and hatching ability at collecting day.

Group Control 0.04 0.08
Fertility, % 48.46c (220/454) 66.59a (301/452) 57.93b (263/454)
Hatching, % 43.18b (95/220) 58.80a (177/301) 40.30b (106/263)
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a–c

Different superscript letters within rows indicate significant difference (P < 0.05).

DISCUSSION

To our knowledge, this study is the first report on the effects of phosphorus and vitamin B12 supplementation in semen extender on Thai native rooster. Our results indicated that 0.04% phosphorus and vitamin B12 supplementation in semen extenders increased sperm's motility, kinetic parameters, sperm characteristics and fertility. This finding may improve the knowledge in increasing chilled rooster sperm quality and fertility through the potential effect of phosphorus and vitamin B12 supplementation in semen extender.

Effect of Phosphorus and Vitamin B12 Supplementation on Sperm Quality

Phosphorus provides cellular energy metabolism and is a substate for ATP cycle (Cunningham, 2002). It is also the phospholipid component in spermatozoa, which is important for sperm function (Gulaya et al., 2001). Vitamin B12 is a cofactor of methylmalonyl-CoA that is used in citric acid cycle and gluconeogenesis involving energy and glucose metabolism (Kennedy et al., 1990). This study found that all concentrations of phosphorus and vitamin B12 supplementation in semen extender improved the sperm total motility, progressive motility, and sperm kinetics. Similarly, Suwimonteerabutr et al. (2020) found that boar's semen quality was improved by butaphosphan and cyanocobalamin supplementation in chilled semen extender. They reported that 0.3% butaphosphan and cyanocobalamin supplementation increased total motility by 6.9% and prolonged sperm lifespan. Furthermore, the use of phosphatidylcholine supplementation as phosphorus in BPSE reduced the damaging effects of lipid peroxidation or enzymatic degradation during storage by providing exogenous phospholipids to sperm membranes (Long and Conn, 2012). In the present work, VCL value in the 0.04% supplementation group was the highest among the control and treatment groups (P < 0.05). VCL is an outstanding predictor and is correlated with the rate of fertilization in males (Farrel et al., 1998; Larsen et al., 2000).

During storage, live spermatozoa are reduced due to high oxidative stress and low energy support (Blesbois et al., 1999). ROS is caused by oxidative injury to sperm, which negatively affects sperm quantity (Amidi et al., 2016) and decreases fertilization capacity (Parodi, 2014). Sperm plasma membrane in rooster contains high polyunsaturated fatty acids, causing high sensitivity with lipid peroxidation due to the ROS on plasma membrane (Cerolini et al., 1997; Breque et al., 2003; Eslami et al., 2016). Vitamin B12 increases the antioxidant status as indicated by the amplified glutathione peroxidase activities, which provide protection against lipid peroxidation and decreased levels of ROS (Banihani, 2017). A combination of phosphorus and vitamin B12 was applied to reduced ROS in chilled and frozen semen in many species (Hu et al., 2011; Hamedani et al., 2013; Hosseinabadi et al., 2020; Bustani and Baiee, 2021). A previous study found that supplementation with vitamin B12 in boar's chilled semen increases semen quality and lifetime (Suwimonteerabutr et al., 2020). Moreover, adding vitamin B12 to the extenders improved the frozen quality, elevated the motility percentage of sperm cells, and improved the movement characteristics, viability, and plasma membrane integrity of bull semen (Hu et al., 2011; Bustani and Baiee, 2021). All these previous studies are consistent with the present study in rooster. We found that chilled rooster semen with all concentrations of phosphorus and vitamin B12 supplementation, especially 0.04%, had a higher percentage of velocity and kinetic parameters of spermatozoa than the control group in all days after collection. Therefore, the beneficial effects of phosphorus and vitamin B12 supplementation on rooster semen quality were increased energy and improved velocity, kinetic parameters, and lifespan in rooster semen.

This study also found that sperm viability and membrane integrity were increased by phosphorus and vitamin B12 supplementation in semen extenders for Thai native roosters. Our results showed that 0.04% phosphorus and vitamin B12 supplementation increased sperm viability by 6.1%, mitochondrial activity by 6.2%, membrane integrity by 7.1%, and acrosome integrity by 1.5% in semen extender. In addition, the quality of Thai native rooster semen with 0.04% phosphorus and vitamin B12 supplementation was better than that of the control group. Previous studies also reported the effect of phosphorus and vitamin B12 supplementation on sperm quality and fertilization in many species. In bulls, 2.50 mg/mL vitamin B12 supplementation increased progressive sperm motility and plasma membrane viability (Hu et al., 2009). In boars, supplementation of 0.5 and 1.0 µg vitamin B12 in extender increased progressive sperm motility and plasma membrane viability (Mello et al., 2013), and 0.3% phosphorus and vitamin B12 supplementation in boar semen extender increased sperm quality (Suwimonteerabutr et al., 2020). In rams, supplementation of 2.0 mg/mL vitamin B12 in the extender improved sperm motility, viability, and plasma membrane viability (Hamedani et al., 2013). Moreover, Hosseinabadi et al. (2020) found that 2 mg/mL vitamin B12 supplementation in frozen semen extender improved the motility and viability of human spermatozoa. Therefore, the data suggested that phosphorus and vitamin B12 supplementation in semen extenders increased the semen quality of Thai native roosters.

High concentrations of phosphorus and vitamin B12 supplementation in semen extenders have an adverse effect on semen which is reported in many species. In boar, Suwimonteerabutr et al. (2020) found that semen with 0.4 and 0.5% of phosphorus and vitamin B12 supplementation in semen extenders negatively affected semen quality and life span. Moreover, the high concentration of vitamin B12 supplementation also reduced cow semen quality (Hu et al., 2011). The present study found that semen with more than 0.06% of phosphorus and vitamin B12 supplementation in semen extenders did not improve semen quality when compared with the control group. The mechanism of phosphorus and vitamin B12 supplementation having an adverse effect on semen quality is still not clear. Excessive use of antioxidant supplements led to heightened cell mortality, as antioxidants are indiscriminate in their ability to distinguish between beneficial and harmful radicals. When antioxidant supplementation is excessively high, it acts as a prooxidant, elevating oxidative stress levels and disrupting the delicate equilibrium between the formation and neutralization of reactive oxygen species (Poljsak et al., 2013)

Effect of Different Concentrations of Phosphorus and Vitamin B12 Supplementation and MDA

As the results of MDA concentration of Thai native rooster semen with 0.04% phosphorus and vitamin B12 supplementation tended to be lower than that of the control group and other experimental groups on day after collection but not significantly difference, it means that 0.04% supplementation group was the most proper concentration for having ability to reduce lipid peroxidation during cooled storage at 4°C. About the importance on level of antioxidants, 0.1% supplementation group which was the highest phosphorus and vitamin B12 concentration was given the highest MDA concentration among groups on day after collection. According to Amini et al. (2015) previously reported that high concentration of vitamin C supplementation in the extender influenced in a decreased activity of antioxidant enzymes such as catalase and glutathione peroxidase as well as an increased production of MDA on rooster post-thawed sperm quality. Long and Kramer (2003) also reported that the supplementation of vitamin E did not reduce lipid peroxidation in turkey semen during cooled storage at 4°C.

Effect of Different Concentrations of Phosphorus and Vitamin B12 Supplementation on Fertility Ability

Sperm characteristics including viability, plasma membrane integrity, and mitochondria activity are important factors contributing to sperm fertilization potential (Blesbois and Brillard, 2007; Shabani et al., 2021). The fertility ability of rooster semen was evaluated to determine its quality. In this study, the 0.04% supplementation group had the highest fertility, which was in accordance with the result of semen quality. However, the fertility in the present work was lower than that in other studies on frozen semen (Chuaychu-noo et al., 2017; Thananurak et al., 2020a,b); this phenomenon can be attributed to the single round of insemination. Additionally, the lipid profile of sperm cells differs by breed (Mussa et al., 2021). Thai native chicken has lower MDA concentration than commercial chicken breed, leading to low sperm quality for the former. Conversely, one report found no difference in fertility between 2 breeds, Pradu Hang dam and Rhode Island Red (Chuaychu-noo et al., 2017). Therefore, the fertility of Thai native rooster should be further explored in the future.

CONCLUSIONS

The use of phosphorus and vitamin B12 supplementation in semen extenders improved sperm motility, sperm kinetics, viability, mitochondrial activity, acrosome integrity, and membrane integrity. Sperm extender supplementation with phosphorus and vitamin B12 supplementation provided energy and protection for Thai native rooster sperm during cooled storage.

ACKNOWLEDGMENTS

This research was funded by National Research Council of Thailand (NRCT) (N41A640105) and Faculty of Veterinary Science Research Fund, 2021, Chulalongkorn University, Bangkok, Thailand (RES_65_068_31_ 013).

DISCLOSURES

This manuscript has not been published or submitted for publication elsewhere. The content does not impose any conflict, and all the authors agree with the manuscript's content.

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