Research Article

The Effect of Glycerol and beta Carotene Supplementation in Tris–Egg Yolk Extender on Swamp Buffalo (Bubalus bubalis) Semen Quality

Rini Elisia1,6*, Maiyontoni1, Tuti Lestari2, Fajri Maulana3, Roni Jarlis4, Romi Andika5

1Animal Husbandry Study Program, Faculty of Mathematics and Natural Sciences, Universitas Negeri Padang, Indonesia; 2Department of Natural Science Education, Faculty of Mathematics and Natural Sciences, Universitas Negeri Padang; 3Department of Agricultural Industrial Technology, Study Program of Animal Feed Technology, Politeknik Negeri Tanah Laut, Tanah Laut 70815, Indonesia; 4Department of Agroindustry, Study Program of Agribusiness, Faculty of Mathematics and Natural Sciences, Universitas Negeri Padang; 5Doctoral Student, Department of Animal Feed and Technology, Faculty of Animal Science, Universitas Andalas, Padang 25163, Indonesia; 6Doctoral Student, Animal Science Study Program, Faculty of Animal Science, Universitas Andalas, Padang 25163, Indonesia.

Received | September 07, 2025; Accepted | November 25, 2025; Published | May 06, 2026

*Correspondence | Rini Elisia, Animal Husbandry Study Program, Faculty of Mathematics and Natural Sciences, Universitas Negeri Padang, Indonesia; Email: [email protected]

Citation | Elisia R, Maiyontoni, Lestari T, Maulana F, Jarlis R, Andika R (2026). The effect of glycerol and beta carotene supplementation in tris–egg yolk extender on swamp buffalo (Bubalus bubalis) semen quality. J. Anim. Health Prod. 14(2): 733-740.

DOI | https://dx.doi.org/10.17582/journal.jahp/2026/14.2.733.740

ISSN (Online) | 2308-2801

Copyright: 2026 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Swamp buffalo is one of the most important livestock in Southeast Asia, particularly in Indonesia, where it is utilized as a source of meat, draft power and cultural heritage (Suhaimi et al., 2015). The advantages of swamp buffalo include good adaptability to wet environments and marginal lands, high tolerance to hot and humid climates and the ability to utilize agricultural by-products with high crude fiber and low nutritional value such as rice straw, corn stover, corn cobs and cocoa pods (Firmansyah et al., 2023).

The development of the swamp buffalo population in Indonesia faces several challenges, one of which is its low reproductive productivity. Low reproductive performance limits the availability of superior breeding stock, making it difficult to improve the population of swamp buffalo (Yani et al., 2025). Artificial insemination (AI) is a reproductive technology that can accelerate genetic improvement and increase livestock populations; however, its success is highly dependent on the quality of frozen semen used (Afrijon et al., 2023). Therefore, improving semen quality through cryopreservation techniques has become an urgent need to support the development of swamp buffalo populations and strengthen food security.

Cryopreservation is the main method for producing frozen semen for AI programs; however, the cooling and freezing processes often cause structural and functional damage to spermatozoa (Moagi et al., 2025). A decline in motility and viability, along with an increase in abnormality of spermatozoa during cooling and freezing, is commonly observed (Imani et al., 2023). These conditions reduce the success rate of AI in swamp buffalo. The formation of ice crystals and oxidative stress can damage sperm cell membranes during freezing and cooling (Herbowo et al., 2020). Such structural and functional damage results in low fertility after insemination in the field, highlighting the need for strategies to improve extenders and minimize losses during frozen semen production in swamp buffalo.

Glycerol is a permeable cryoprotectant widely used in semen extenders because of its ability to protect spermatozoa from freezing-induced damage (Ariantie et al., 2013). However, the use of glycerol requires optimization since concentrations that are too low provide insufficient protection, while excessively high levels can be toxic to spermatozoa (Nalley et al., 2011; Herbowo et al., 2020).

The addition of antioxidants also plays a crucial role in maintaining semen quality during cryopreservation. Beta-carotene is a natural antioxidant capable of reducing oxidative stress by neutralizing free radicals, thereby protecting the sperm cell membrane (Zareba et al., 2013). Oxidative damage during cooling and freezing is one of the major causes of fertility decline (Herbowo et al., 2020).

Most studies on semen cryopreservation have focused on cattle, while research on swamp buffalo remains limited, even though improving semen quality is essential for the development of swamp buffalo. The development of swamp buffalo aims to preserve local genetic resources, enhance the efficiency of AI programs and improve the livelihoods of farmers who depend on buffalo husbandry (Afrijon et al., 2023).

Based on the above, this study was conducted to evaluate the optimal concentrations of glycerol and beta-carotene in Tris–egg yolk extender to maintain the quality of swamp buffalo spermatozoa. The urgency of this research lies in its contribution to producing higher-quality frozen semen, which can increase the success rate of artificial insemination and support programs to improve the swamp buffalo population.

MATERIALS AND METHODS

Animals and collection of semen

In this study, two swamp buffalo bulls aged 4 years, with a body weight of 600–700 kg, were used as semen donors. The animals were selected from breeding male swamp buffaloes at the Tuah Sakato Artificial Insemination Center, Payakumbuh City, West Sumatra, Indonesia. Semen was collected using an artificial vagina and only ejaculates with mass motility ≥70% were used in this study (Indonesian National Standard, 2017).

Experimental semen extenders

The basic extender used was Tris–egg yolk, prepared under sterile conditions and pre-warmed (±37 °C) before use. The composition of Tris–egg yolk consisted of a Tris–citric acid–glucose solution supplemented with 10% (v/v) fresh egg yolk as a source of membrane-protective lipoproteins and antibiotics according to laboratory SOP. The supplementation treatments were arranged into eight groups as follows: A (control; Tris–egg yolk without additives), B (glycerol 2%), C (glycerol 4%), D (glycerol 6%), E (glycerol 8%), F (beta-carotene 0.1%), G (beta-carotene 0.2%) and H (beta-carotene 0.3%).

Fresh semen that met the eligibility criteria was then diluted with each extender at a ratio resulting in a final concentration of approximately 20×10⁶ spermatozoa per 0.25 ml straw (≈80×10⁶/ml). Extender addition was carried out gradually (2–3 fractions) under warm conditions (±37 °C) with gentle inversion to minimize cold shock and osmotic stress. All procedures were conducted aseptically using pre-warmed glassware and protected from direct light exposure, particularly for treatments containing beta-carotene.

Research procedure

Donor animals

The donor animals used in this study were two healthy male swamp buffaloes. The swamp buffaloes were given an adaptation period of 1–2 weeks and ensured to be free from reproductive disease symptoms. The buffaloes were lightly fasted for 6–8 hours before the semen collection process, but still provided free access to drinking water ad libitum.

Semen collection

Semen collection was carried out using an artificial vagina (AV) which was prepared with the water temperature inside maintained at 42–45 °C and the tip of the liner given a thin lubrication to facilitate ejaculation. The collection process could be assisted with a teaser and usually performed 2–3 times until an ejaculate with qualified quality was obtained. The semen released was immediately collected into a pre-warmed collection tube at 37 °C, then the tube was tightly closed to prevent contamination of the semen.

Fresh semen evaluation

Fresh semen evaluation was carried out immediately after collection which included macroscopic and microscopic examinations. Macroscopically, semen was observed based on volume, color, odor and pH. Microscopically, semen quality was examined through several parameters namely concentration, mass movement and motility.

Preparation of Tris–egg yolk extender

The semen extender used in this study was Tris–egg yolk which was prepared in a sterile manner with the basic composition consisting of Tris, citric acid, glucose and antibiotics. The basic solution was added with fresh egg yolk as much as 10% (v/v) as a source of lipoproteins and phospholipids that function to protect the plasma membrane of spermatozoa, glycerol with concentrations according to the treatment that acts as a cryoprotectant, beta-carotene as antioxidant with concentrations according to the treatment.

The extender solution was then filtered to ensure the clarity and cleanliness of the medium and preheated until it reached a temperature of 37 °C before being used to dilute the semen to prevent the occurrence of cold shock in the spermatozoa.

Buffalo semen dilution process

At the dilution stage, semen was aseptically divided into eight treatment groups, namely: A (control; without addition), B (glycerol 2%), C (glycerol 4%), D (glycerol 6%), E (glycerol 8%), F (beta-carotene 0.1%), G (beta-carotene 0.2%) and H (beta-carotene 0.3%). Each part was then diluted using extender preheated at ±37 °C with the aim of reaching a final density of about 20×10⁶ spermatozoa per 0.25 ml straw (equivalent to 80×10⁶/ml).

The calculation of the extender volume was based on the semen concentration, then the addition of the extender was carried out gradually (2–3 fractional additions) while the tube was gently inverted to ensure it was evenly mixed and to minimize cold shock as well as osmotic stress.

Cooling stage

The cooling stage was carried out by lowering the temperature of the diluted semen from room temperature to reach 4–5 °C gradually. This process lasted for approximately 1.5–2 hours with a cooling rate of about 0.2–0.3 °C per minute. Gradual cooling was very important to prevent cold shock, namely damage to the spermatozoa plasma membrane due to excessively rapid temperature changes. During the cooling process, semen was placed on a rack or special box in the refrigerator to maintain the stability of position and temperature.

Equilibration at 4–5 °C

After the semen temperature reached 4–5 °C, samples from each treatment were incubated at that temperature to undergo the equilibration process. The equilibration time was determined according to the research design, namely 1 hour, 3 hours and 5 hours. During the equilibration process, semen tubes were stored tightly closed on the storage rack and kept away from direct light exposure to prevent oxidative stress that could reduce spermatozoa quality.

Post-equilibration semen evaluation

Semen examination was carried out at the end of each equilibration time, namely 1 hour, 3 hours and 5 hours. The parameters observed included viability, motility and abnormalities of swamp buffalo semen post-equilibration. All observations were carried out on glass slides preheated to a temperature of 37 °C using a warm stage, so that examination conditions were close to physiological temperature.

Packaging (strawing)

The semen packaging process was carried out in a cold working room at about 5 °C to maintain the stability of spermatozoa quality. The diluted semen was filled into 0.25 ml straws with a headspace of about 4–5 mm at the top to prevent excessive pressure during freezing. The straws were then sealed using heat or PVA balls according to laboratory standards so that they were completely tightly closed and free of contamination.

Freezing

The freezing stage was carried out using the liquid nitrogen vapor method to maintain spermatozoa viability and integrity. Straws containing semen were arranged on a rack at a distance of about 4–5 cm above the surface of liquid nitrogen (LN₂) for 10–15 minutes so that a gradual cooling process occurred through nitrogen vapor exposure. Next, the straws were immediately immersed directly into LN₂ at a temperature of (−196 °C), for long-term storage.

Thawing process

The thawing process was carried out by immersing straws containing frozen semen into water at 37 °C for 30–60 seconds. After the thawing time was completed, the straws were immediately lifted and the outer part was dried with clean tissue to prevent water contamination. The tip of the straw was cut with sterile scissors and the straw contents were released into a pre-warmed tube at 37 °C to maintain semen temperature stability before further evaluation of post-thawing quality was carried out.

Post-thawing semen quality evaluation

The evaluation of post-thaw semen quality was carried out at a maximum of ≤10 minutes after the thawing process to obtain representative results. The main parameters observed included viability, motility, abnormalities and progressive sperm motility post-thawing.

Performance parameters

The performance parameters evaluated in this study consisted of three stages of semen quality assessment in swamp buffalo (Bubalus bubalis). First, the quality of fresh semen was examined to determine the initial characteristics of the ejaculate prior to further processing. Second, post-equilibration semen quality was assessed by measuring sperm viability, motility, and morphological abnormalities to evaluate sperm resistance during the cooling phase. Third, post-thaw semen quality was evaluated based on sperm viability, motility, abnormalities, and progressive motility, which served to determine the effectiveness of the treatments in maintaining sperm function following the freezing-thawing process.

Data analysis

Each data set was analyzed using ANOVA according to the Randomized Block Design (RBD) method. If the calculated F value was greater than the F table at the 5% significance level, the data analysis was continued using Duncan’s Multiple Range Test (DMRT) based on Steel and Torrie (1995).

RESULTS AND DISCUSSION

Fresh semen quality of buffalo

The evaluation results showed that macroscopically, the fresh semen of swamp buffalo had an average semen volume of 2.26±1.15 ml (Table 1). This finding is consistent with that reported by Kumar et al. (2023), where buffalo bulls produced fresh semen of 2.82 ± 1.45 mL. However, it was lower than the semen volume of Murrah buffalo, which was 4.40±0.18 ml (Isnaini et al., 2019). Factors determining the semen volume of swamp buffalo include age, body weight, and the quantity of feed provided. One advantage of buffalo semen is that its sperm concentration is relatively higher than that of cattle, so although semen volume is lower, it remains highly potential for use in artificial insemination programs (Perumal, 2014).

 

Table 1: The fresh semen quality of swamp buffalo used in this study.

Parameters	Test results
Volume (ml)	2.26±1.15
Color	Creamy
Odor	Characteristic
pH	6.54±0.08
Concentration (106/ml)	1.182±386.91
Mass motility	++ (good)
Motility (%)	71±2.00

 

The semen color obtained in this study was creamy with a characteristic odor (Table 1), indicating that the fresh swamp buffalo semen originated from normal accessory gland secretions without contamination or infection. Buffalo semen color generally varies between milky white to yellowish cream, influenced by sperm concentration and semen plasma composition (Andrabi, 2009). The semen pH value obtained was 6.54 ± 0.08 (Table 1), which falls within the normal pH range of swamp buffalo semen. Seasons do not affect buffalo semen pH, which ranges from 6.9 ± 0.0 to 7.0 ± 0.0 (Koonjaenak et al., 2007).

The semen concentration of swamp buffalo in this study was 1.182 ± 386.91 × 10⁶/ml (Table 1). This value is still within the semen concentration range of buffalo reported in several references, ranging from 800 × 10⁶ to 1,500 × 10⁶ spermatozoa/ml, depending on age, nutritional status and ejaculation frequency of the bull (Sansone et al., 2000; Perumal, 2014). This concentration is relatively high and supports the potential use of buffalo semen in artificial insemination programs, since an adequate number of spermatozoa is crucial to ensure fertility.

The mass motility of fresh swamp buffalo semen in this study was categorized as good (Table 1). This indicates that the spermatozoa had synchronized and strong forward thrust. Mass motility is an important indicator for visually assessing sperm quality, where strong and wavy swirling movements reflect good sperm metabolism and motility (Kumar et al., 2023).

Individual motility of fresh swamp buffalo semen obtained in this study was 71 ± 2.00% (Table 1). This value is relatively high and meets the quality standard of fresh semen for cryopreservation, in which a minimum motility of 70% is required (Iqbal et al., 2016; INS, 2017). Sperm motility is strongly influenced by plasma membrane integrity and mitochondrial activity, both of which determine the ability of sperm to move progressively and reach the oocyte.

Overall, the evaluation results of fresh swamp buffalo semen in this study fall within the normal range, indicating that the bulls used had good reproductive potential. The quality of fresh swamp buffalo semen is highly important since the success of cryopreservation and artificial insemination largely depends on the semen quality prior to freezing.

Effect of glycerol and beta-carotene on swamp buffalo sperm quality post-equilibration

The addition of glycerol and beta-carotene in tris egg yolk diluent had a significant effect (p < 0.05) on the viability, motility and abnormality of swamp buffalo semen post-equilibration. The viability of swamp buffalo spermatozoa post-equilibration ranged from 51.27 ± 1.96% to 65.57 ± 1.48% (Table 2). The viability of spermatozoa in the control group (Treatment A) was 51.27 ± 1.96% and the value increased with the addition of glycerol, as observed in Treatment E (8% glycerol) at 65.57 ± 1.48%, Treatment D (6% glycerol) at 63.15 ± 1.51% and Treatment C (4% glycerol) at 61.97 ± 1.16%. This indicates that glycerol supplementation protects spermatozoa during the equilibration process by penetrating the cell, replacing part of the intracellular water, and preventing the formation of ice crystals that can damage the plasma membrane (Watson, 2000; Perumal, 2014). The higher the glycerol concentration, the more optimal the protection of the cell membrane, although at excessively high concentrations (>10%) glycerol has been reported to cause toxicity (Andrabi, 2009).

The viability of swamp buffalo spermatozoa post-equilibration with beta-carotene supplementation also showed a positive effect, although not as high as with glycerol. Viability increased from 53.48 ± 0.99% (F: Beta-carotene 0.1%) to 58.68 ± 1.87% (H: Beta-carotene 0.3%) (Table 2). Beta-carotene acts as a natural antioxidant that neutralizes free radicals, thereby reducing lipid peroxidation in spermatozoa membranes, which are highly susceptible to oxidative stress (Zareba et al., 2013). Supplementation with 0.3% beta-carotene (Treatment H) yielded the best results among the antioxidant treatments, although still lower than Treatment E with 8% glycerol.

The motility of swamp buffalo semen post-equilibration ranged from 64.41 ± 0.88% to 78.12 ± 1.76% (Table 2). The addition of glycerol and beta-carotene in tris egg yolk diluent had a significant effect on spermatozoa motility post-equilibration. The control (Treatment A) had the lowest motility (64.41%). This indicates that without cryoprotectant or antioxidant supplementation, spermatozoa are more vulnerable to damage during cooling. Glycerol plays an important role as a permeable cryoprotectant that enters the cell, binds water, lowers the freezing point and prevents ice crystal formation that could damage the spermatozoa membrane. Without cryoprotectants in the semen diluent, spermatozoa are highly susceptible to membrane damage, protein denaturation and acrosome dysfunction during cooling (Medeiros et al., 2002). Moreover, without antioxidant protection, the accumulation of free radicals during cooling induces lipid peroxidation, DNA damage and spermatozoa abnormalities (Agarwal et al., 2014). The use of glycerol at an optimal concentration helps maintain plasma membrane integrity and preserves spermatozoa motility after equilibration.

 

Table 2: The effect of glycerol and beta-carotene supplementation in a Tris–egg yolk diluent on the quality of swamp buffalo spermatozoa post-equilibration.

Treatment	Post- equilibration (%)
	Viability	Motility	Abnormality
A (Control)	51.27±1.96	64.41±0.88	45.63±1.57
B (Glycerol 2%)	59.53±2.23	73.03±1.61	41.86±1.11
C (Glycerol 4%)	61.97±1.16	73.45±2.69	41.12±1.31
D (Glycerol 6%)	63.15±1.51	73.77±1.48	39.46±1.56
E (Glycerol 8%)	65.57±1.48	78.12±1.76	35.58±1.80
F (Beta-carotene 0.1%)	53.48±0.99	68.18±1.09	42.35±0.63
G (Beta-carotene 0.2%)	57.49±2.21	70.98±2.34	42.12±0.95
H (Beta-carotene 0.3%)	58.68±1.87	69.64±2.06	41.89±1.97
SEM	0.70	0.76	0.65
P-Value	*	*	*

 

Description: SEM: Standard error of the mean; *: Significant.

 

The addition of beta-carotene also improved motility compared with the control, although the effect was not as strong as glycerol. The best motility was obtained at a concentration of 0.2% (70.98%), indicating the role of beta-carotene as a strong antioxidant in scavenging free radicals and preventing lipid peroxidation in spermatozoa membranes (Castro et al., 2018). However, a higher concentration of beta-carotene (Treatment H, 0.3%) showed a slight decrease in motility, which may be attributed to the pro-oxidant properties of beta-carotene when used at excessive levels (El-Agamey et al., 2004). Overall, glycerol proved to provide stronger protection for spermatozoa motility through cryoprotective mechanisms, whereas beta-carotene served as an additional protector by reducing oxidative stress occurring during cryopreservation.

The abnormality of swamp buffalo semen post-equilibration ranged from 35.58 ± 1.80% to 45.63 ± 1.57% (Table 2). The results showed that the addition of glycerol and beta-carotene in tris egg yolk diluent influenced the abnormality level of swamp buffalo spermatozoa post-equilibration. In the control group (A), spermatozoa abnormality reached 45.63 ± 1.57%, which was relatively higher compared with all other treatments. This indicates that without cryoprotectant (glycerol) or antioxidant (beta-carotene) supplementation, the structure of spermatozoa is more prone to damage during the cooling process.

Effect of glycerol and beta-carotene on swamp buffalo sperm quality post-thaw

The addition of glycerol and beta-carotene in tris egg yolk diluent had a significant effect (p < 0.05) on the viability, motility, abnormality and progressive sperm motility of swamp buffalo semen post-thawing. Post-thawing viability ranged from 51.27 ± 1.76% to 65.57 ± 2.39% (Table 3). Spermatozoa in the control group (Treatment A) had the lowest viability (51.27%), while the gradual addition of glycerol increased viability, reaching the highest value at 8% glycerol (Treatment E) with 65.57%. This indicates that glycerol is effective as a permeable cryoprotectant that enters the cell and replaces intracellular water, thereby preventing ice crystal formation and maintaining plasma membrane integrity during freezing and thawing (Andrabi, 2009; Andrabi et al., 2008). The addition of beta-carotene also increased viability compared to the control, particularly at 0.3% (Treatment H) with 58.68%, although the effect was not as strong as glycerol. The role of beta-carotene is more antioxidative, preventing membrane damage caused by lipid peroxidation by reactive oxygen species (ROS) during cryopreservation (Castro et al., 2018).

Post-thawing motility ranged from 47.02 ± 2.66% to 64.43 ± 1.77% (Table 3). Sperm motility after thawing also increased significantly with glycerol supplementation. The lowest motility value was found in the control (Treatment A) at 47.02%, while the highest motility was observed at 8% glycerol (Treatment E) with 64.43%. This confirms the protective role of glycerol in maintaining membrane function and enzymatic systems responsible for sperm movement. Beta-carotene supplementation showed moderate improvement, with the best result at 0.3% (Treatment H) with 59.19%. The protective mechanism of beta-carotene comes from its antioxidant properties, which protect sperm mitochondria from oxidative damage, thereby preserving the energy required for motility (Azawi et al., 2012).

Post-thawing abnormality ranged from 18.53 ± 0.49% to 31.28 ± 1.06% (Table 3). The highest sperm abnormality occurred in the control (Treatment A) at 31.28%, while glycerol supplementation significantly reduced abnormalities, with the lowest value observed at 8% glycerol (Treatment E) with 18.53%. This reduction strengthens the evidence that glycerol protects sperm structure from physical damage caused by osmotic stress and ice crystals. Beta-carotene supplementation also reduced abnormalities, although its effect was smaller (27–29%). This reduction is presumably due to the ability of beta-carotene to suppress oxidative damage to sperm membranes, thereby maintaining cell morphology stability (El-Agamey et al., 2004).

Progressive sperm motility ranged from 36.63 ± 1.11% to 52.46 ± 1.05% (Table 3). Progressive motility is an important parameter to assess the fertilization potential of spermatozoa. The results showed that the control (Treatment A) had the lowest value (36.63%), whereas 8% glycerol (Treatment E) gave the highest result (52.46%).

 

Table 3: The effect of glycerol and beta-carotene supplementation in Tris egg yolk diluent on the quality of swamp buffalo spermatozoa post-thawing.

Treatment	Post-Thawing (%)
	Viability	Motility	Abnormality	Progressive sperm motility
A (Control)	51.27±1.76	47.02±2.66	31.28±1.06	36.63 ± 1.11
B (Glycerol 2%)	59.53±1.87	61.67±0.84	26.28±0.90	41.77 ± 1.85
C (Glycerol 4%)	61.97±2.84	61.78±1.16	26.12±1.51	42.91 ± 1.21
D (Glycerol 6%)	63.15±2.28	62.59±1.46	24.50±0.94	43.87 ± 1.05
E (Glycerol 8%)	65.57±2.39	64.43±1.77	18.53±0.49	52.46 ± 1.63
F (Beta-carotene 0.1%)	53.48±2.21	48.54±1.71	29.73±0.66	39.19 ± 0.75
G (Beta-carotene 0.2%)	57.49±2.67	54.32±1.34	28.61±1.13	41.25 ± 1.33
H (Beta-carotene 0.3%)	58.68±1.92	59.19±1.09	27.65±0.98	41.66 ± 0.47
SEM	1.26	0.86	0.49	0.67
P-Value	*	*	*	*

 

Description: SEM: Standard error of the mean; *: Significant.

 

The increase in progressive motility reflects good structural and functional membrane quality, enabling more directed sperm movement. The addition of beta-carotene provided a slight improvement, with the best value at 0.3% (Treatment H) with 41.66%. This suggests that although beta-carotene helps maintain cell function through its antioxidant mechanism, its role is not as strong as glycerol in supporting progressive motility of spermatozoa post-thawing.

CONCLUSIONS AND RECOMMENDATIONS

The conclusion of this study is that the addition of 8% glycerol in tris egg yolk extender for swamp buffalo semen was the best treatment, resulting in 65.57% post-equilibration viability, 78.12% post-equilibration motility and 35.58% post-equilibration abnormality, while post-thawing values were 61.63% viability, 64.43% motility, 31.28% abnormality and 31.28% post-thaw progressive sperm motility.

ACKNOWLEDGMENTS

This research was funded by the Research and Community Service Institute (LPPM) of Universitas Negeri Padang under contract number 1297/UN35.15/LT/2023. The authors would like to express their gratitude for the support and facilitation provided.

NOVELTY STATEMENT

1. This study is the first comprehensive investigation to evaluate the effectiveness of combining glycerol and beta-carotene in Tris–egg yolk extender on the quality of swamp buffalo spermatozoa at both the post-equilibration and post-thawing stages.
2. The study introduces the optimization of glycerol concentration as a permeable cryoprotectant, serving as a physical protector of spermatozoa against cryodamage, while also identifying the threshold level to prevent toxicity in swamp buffalo semen.
3. The addition of beta-carotene as a natural antioxidant in the swamp buffalo semen extender represents a novel approach to reducing oxidative stress caused by free radical formation during cryopreservation, thereby potentially enhancing post-thaw viability and motility of spermatozoa.
4. The findings of this study provide a scientific contribution to the field of swamp buffalo reproductive cryobiology, thereby supporting programs for the conservation of local genetic resources and improving the success of artificial insemination in swamp buffaloes.

AUTHOR’S CONTRIBUTION

RE designed the study, developed the methodology, curated the data and prepared the original draft. M performed formal analysis, software processing and validation; TL supervised the research, provided resources and secured funding. FM managed the project and contributed to writing, review and editing. RJ conducted the investigation and prepared visualizations. RA collected data, conducted laboratory analyses and contributed to writing review and editing.

Ethical approval

This study was conducted at the Tuah Sakato Artificial Insemination Center (AIC), Payakumbuh City, West Sumatra, Indonesia, in accordance with the standard operating procedures of SNI ISO 9001:2015 and SNI ISO 37001:2016. The research was supervised by a veterinarian and approved by the Ethics Committee regarding the responsible use of breeding bulls for fresh semen collection. This study complied with the principles of animal experimentation as stipulated in the Decree of the Minister of Agriculture of the Republic of Indonesia No. 306/KPTS/TN.330/4/1994 and Law of the Republic of Indonesia No. 18 of 2009 on Animal Husbandry and Animal Health, as amended by Law No. 41 of 2014.

Generative AI and AI-assisted technology statement

The authors declare that ChatGPT (OpenAI, San Francisco, CA, USA) was used to assist in the initial translation of the manuscript from Indonesian to English. All outputs were reviewed, edited, and verified by the authors to ensure accuracy and originality.

Conflict of interests

The authors have declared no conflict of interest.

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