Research Article

Impact of Pre-Freezing Duration on Buck Semen Quality in Eppendorf Tubes

Lalu Ahmad Zaenuri*, Enny Yuliani

Faculty of Animal Science University of Mataram, Majapahit Street, No. 62, Mataram Lombok, West Nusa Tenggara, Indonesia.

Received | December 27, 2024; Accepted | January 14, 2025; Published | February 08, 2025

*Correspondence | Lalu Ahmad Zaenuri, Faculty of Animal Science University of Mataram, Majapahit Street, No. 62, Mataram Lombok, West Nusa Tenggara, Indonesia; Email: [email protected]

Citation | Zaenuri LA, Yuliani E (2025). Impact of Pre-freezing duration on buck semen quality in eppendorf tubes. Adv. Anim. Vet. Sci. 13(3): 484-491.

DOI | https://dx.doi.org/10.17582/journal.aavs/2025/13.3.484.491

ISSN (Online) | 2307-8316; ISSN (Print) | 2309-3331

Copyright: 2025 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/).

INTRODUCTON

Semen preservation research has been conducted for an extended period, and its benefits are currently being validated, particularly in enhancing reproductive efficiency and livestock productivity. Chunrong et al. (2019) noted that semen preservation has become an integral component of the modern livestock industry. This technique is well-known in animal breeding, including for goat. Crossbreeding semen from superior exotic goat breeds with local goats enhances genetic quality and productivity. (Myagmarsuren et al., 2016). Agarwal et al. (2004), Tash et al. (2003) and Yimer et al. (2015) stated that semen preservation facilitates the distribution of superior breeds, which is essential for rapid genetic improvement and increased production of local livestock. Ultimately, this contributes to meeting the ever-growing human demand for livestock products. Chunrong et al. (2019) further explained that semen preservation not only directly supports artificial insemination (AI) programs but also significantly reduces the costs associated with maintaining breeding studs. Consequently, semen preservation remains a compelling area of research that continues to attract the attention of many researchers (Chunrong et al., 2019; Walters et al., 2009).

Semen cryopreservation, particularly in bucks and cattle, has recently benefited from the adoption of modern techniques and equipment, which necessitate significant financial investment (Chunrong et al., 2019). The entire process is facilitated by automated tools and machinery, enhancing efficiency and ensuring that the quality of the frozen semen produced is optimal (Arman, 2014). Nevertheless, post-thaw sperm motility remains approximately 50%, primarily due to damage sustained by sperm to the plasma membrane, nucleus, mitochondria, acrosome, and overall morphology during the freezing process (Walter et al., 2009). In contrast, the utilization of artificial insemination (AI) technology has been steadily increasing; however, not all institutions, particularly those involved in goat farming, possess the modern facilities required for the production of frozen semen.

Sperm cryopreservation has experienced significant advancements regarding the techniques utilized for freezing, the speeds at which samples are frozen and thawed, and the preservation media employed to maintain sperm functionality and DNA integrity (Hungerford et al., 2022). References concerning the freezing of buck semen are less abunandt compared to those available for bovine semen (Morrell et al., 2022). Frozen semen can be stored in a variety of packaging formats, including ampoules, pellets, mini-tubes, and fine straws. Of these options, fine straw packaging is currently the most widely utilized (Ramirez-Reveco et al., 2016).

Unlike contemporary freezing methods, this study was conducted in a small laboratory with very limited resources, focusing on a cost-effective, manual and applicable approach that can be implemented in small laboratories worldwide. Consistent with the findings of Morrell et al. (2022), the buck semen freezing method should be tailored to the specific conditions under which the work is conducted. The objective of this study was to investigate the effects of Eppendorf packaging and the duration of pre-freezing with a manual protocol on the quality of post-thaw Kacang buck semen. The goal is to make semen preservation more accessible to farmers by proposing simplified techniques that can be implemented in small, remote laboratories.

MATERIALS AND METHODS

Kacang Buck

The subject of this study is a local Indonesian buck, specifically the Kacang breed, three years old, and weighing 45 kg. This buck has been confirmed to be fertile, healthy, and free from both internal and external parasites. The buck is housed in a stage cage measuring 2×3 m². Its diet consists of high-quality fresh leaves from the legume tree (Sesbania glandiflora), provided in the morning and afternoon at a quantity equivalent to 15% of its body weight. Access to drinking water is freely available in a plastic bucket container.

Treatment Extenders

The most commonly used extender for semen cryopreservation in Indonesia is Tris-egg yolk (Nisfimawardah et al., 2023). The buffered experimental extender was prepared by dissolving 3.634 g of Tris (hydroxymethyl) aminomethane (Sigma, USA), 2.17 g of citric acid monohydrate (Merck, Germany), 0.50 g of fructose (Merck, Germany), 0.06 g of sodium penicillin G (Wonder, Japan), and 0.1 g of streptomycin (Wonder, Japan) in 100 ml of distilled water to achieve a pH of 7.0, following the protocol by Evans and Maxwell (1987). The prepared extender was stored in a refrigerator until needed. Just before semen collection, the extender was mixed with 2.5% egg yolk (v/v) at room temperature (Evans and Maxwell, 1987). The extender was then divided into three test tubes, each containing 10 ml, and kept at room temperature until use. Additionally, a tank of liquid nitrogen (LN2) was prepared for the preservation process.

Semen Collection, Extended, Freezed and Evaluation

Ten ejaculates were collected from the buck every four days using an artificial vagina (AV) and transported to the laboratory for assessment. Criteria for suitability included a volume of at least 0.8 ml, a wave motion rating of +++, total motility of at least 70%, and a concentration of at least 2.5 x 10^9 sperm per milliliter (Evans and Maxwell, 1987; Menchaca et al., 2005).The concentration of sperm was determined using a hemocytometer. The concentration of sperm per unit volume (ml) was calculated using Thoma haemocytometer (Evans and Maxwell, 1987).

The method to prepare a 3% NaCl solution (Susilawati, 2013). Briefly, by dissolving 1.5 grams of NaCl in 50 mL of distilled water and homogenizing it. Use a micropipette to take 10 μl of fresh semen and drop the semen into a test tube. Mix the fresh semen with 1990 μl of 3% NaCl solution, which means that the semen has been diluted 200×; homogenize the semen and 3% NaCl solution by shaking the tube slowly several times. Take 2 μl of semen sample using a micropipette and drop it on the side of the haemocytometer cover glass; then the sperm are observed using a light microscope with 400× magnification. Count the sperm in 5 large boxes, each box consisting of 16 small boxes, so that the total number of boxes counted is 80 small boxes. The concentration of sperm per ml is the number of sperm counted multiplied by 107.

Fresh semen meeting all criteria was promptly processed by mixing it with an extender at a 1:50 ratio (Menchacha et al., 2005). The liquid semen was then divided into three tubes, each containing 10 ml of diluted semen. These tubes were placed in a pre-equilibrated water jacket to gradually decrease the temperature of the semen by 0.25ºC per minute, reaching 5 ºC within five hours (Arman, 2014; Menchacha et al., 2005).

The addition of 7% (v/v) glycerol was carried out in a refrigerated environment three hours after semen stored in refrigerator (Arman, 2014) and then homogenized by gently shaking the tube. One hour before pre-freezing, each tube containing 10 ml of diluted semen was aliquoted into five Eppendorf tubes, each with a capacity of 1 ml, based on the observation frequency for each treatment, resulting in a total of 15 Eppendorf tubes. This protocol was repeated for the second through tenth semen collections.

The freezing process involves several steps. First, prepare a styrofoam box measuring 24 cm x 22 cm x 20 cm and pour liquid nitrogen into it. Next, use a second styrofoam box measuring 8 cm x 15 cm x 13 cm, placing a fine wire mesh 4 cm from the bottom to allow it to float on the LN2 surface, enabling vapor entry. Then, position an Eppendorf tube containing liquid semen in the second box. Pre-freezing treatments T1, T2 and T3 are conducted for 10, 15, and 20 minutes, respectively, before immersion in liquid nitrogen. Finally, label all treatment tubes and store them in a goblet.

Post-Thaw Evaluation

Thawing was conducted by immersing the Eppendorf tube in water at 50°C for 73 seconds, similar to thawing semen in ampoules (Ramirez-Reveco et al., 2016). The study evaluates sperm quality post-thawing, including motility, viability, and membrane integrity, with observations recorded immediately and every 30 minutes for 120 minutes. The evaluation of total motility, progressive motility, and viability followed the methodology outlined by Maxwell and Salamon (1993). A drop of fresh semen was mixed with 1 ml of extender, placed on a glass slide, and covered with a cover slip. Total motility was subjectively assessed at 200× magnification by estimating the proportion of motile sperm. Progressive motility, defined as sperm exhibiting straight-line movement, was evaluated at 400× magnification with 200 sperm analyzed (Swarna et al., 2022). The total total and progressive motility were quantified across multiple fields of view and expressed as percentages. The percentage of total and progressive motility was calculated by dividing the number of motile sperm by 200 and multiplying by 100% (Swarna et al., 2022; Zaenuri et al., 2023).

The integrity of the plasma membrane was evaluated using a hypo-osmotic solution (HOS) (Alcay et al., 2015) made with fructose and sodium citrate at concentrations of 8.72 g/L and 4.74 g/L, a pH of 8.05, respectively (Susilawati, 2013; Alcay et al., 2015). A 10 µL aliquot of semen was mixed with 200 µL of the HOS solution and incubated at 37°C for 40 minutes (Jeyendran et al., 1984; Susilawati, 2013; Suwimonteerabutr et al., 2020). After incubation, a drop of the semen mixture was placed on a glass slide, covered with a cover slip, and examined under a light microscope (Nikon, Japan) at 400× magnification. Microscope fields were randomly selected for analysis. Two hundred sperm were counted per slide, and the percentage of swollen-tailed sperm was calculated. The percentage of sperm with intact membranes was determined by dividing the number of sperm with circular tails by 200 (Jeyendran et al., 1984; Ahmad et al., 2003; Susilowati et al., 2021; Suwimonteerabutr et al., 2020).

Sperm viability was assessed by preparing smears from each semen treatment. To create a smear, a drop of post-thaw semen was mixed with Eosin-Nigrosin solution (Wonder, Japan). The smear was examined under a light microscope (Nikon, Japan) at 400× magnification. Sperm were classified as viable if they did not absorb the dye, and those that absorbed the dye were considered non-viable.

Data Analysis

Data obtained from the postthawing evaluation results were analyzed using a Completely Randomized Design (CRD) analysis of variance, a 3 x 2 factorial pattern with ten replications (Steel and Torrie, 1993). Data were analyzed using One Way ANOVA for completely randomized design using Co-Stat for windows statistical software (version 6.03). If there were differences between treatments, the analysis was continued with the Duncan test (α = 0.05).

RESULTS AND DISCUSSION

Fresh Semen

This study investigated the free-freezing duration of buck semen in Eppendorf tubes, using semen from one Kacang buck for homogeneity. Semen collection using an artificial vagina (AV) proved to be an effective method for obtaining high-quality semen in this study. The bucks displayed high libido and quickly adapted to ejaculating into the AV when presented with anoestrous does, even in the presence of other males. The average volume and pH of the semen, as well as the concentration, total motility, and viability of the buck sperm from ten collections using the AV, were recorded as 0.89 mL, 6.8, 2.85 x 10^9/mL, 90.2%, and 95%, respectively. This indicates that the semen from the buck meets the criteria for processing into frozen semen (Evans and Maxwell, 1987; Menchacha et al., 2005). Cryopreservation is essential for long-term storage of semen at -196°C while maintaining fertilization rates post-thawing (Lemma, 2011). However, it is important to note that negative effects on total motility, progressive motility, viability, and the integrity of the sperm plasma membrane may occur during the freezing and thawing processes (Watson, 2000). However, it could be noted that the main factor to evaluate quality characteristics of sperm such as motility, plasma membrane integrity, and viability (Amini et al., 2019).

Post-Thaw Semen Quality

Total sperm motility: The total motility of Kacang buck sperm after thawing (Table 1) shows a pattern where longer pre-freezing durations lead to higher total sperm motility. On the other hand, storing the sperm at room temperature for an extended period results in a decrease in total motility. Eppendorf tube packaging for freezing goat semen does not harm spermatozoa quality. A negative impact may stem from other factors. Ugur et al. (2019) propose that damage to sperm could be caused by factors such as hyperosmolarity, ionic imbalance, protein denaturation, generation of reactive oxygen species (ROS), and inadequate energy supply during cooling and freezing processes.

 

Table 1: The impact of pre-freezing duration and room temperature storage time on the total motility of post-thaw buck sperm packed in eppendorf tubes.

Post thawing (minute)	Treatments
	T1	T2	T3
0	51.02 ±2.57a	61.40±2.31b	62.19±3.15b
30	48.10±6.91a	55.70±1.95b	57.20±1.44b
60	37.12±5.18a	44.18±3.26b	49.16±2.11b
90	29.25±4.21a	36.01±3.11b	48.23±3.21c
120	24.31±4.25a	29.11±3.26a	41.19±1.12c

 

Note: different superscripts in the same column indicate significant differences (P < 0.05).

 

The study findings indicate that total motility assessed at 0 minutes post-thawing is relatively high, with all three treatment groups showing motility rates exceeding 50%. However, there was a significant decline in total motility shortly after thawing from 51.02 ± 2.57 to 24.31 ± 4.25 at 120 minutes in T1. This reduction in T1 was statistically significant (P < 0.05) compared to T2 and T3, which had motility rates of 61.40 ± 2.31 to 29.11 ± 3.26 and 62.19 ± 3.15 to 41.19 ± 1.12, respectively. Table 1 shows that frozen semen at T1, T2, and T3 can be maintained respectively for 30, 60, and 120 minutes for AI purposes, as total motility remains above 40% (Standard Nasional Indonesia, 2014). Additionally, the percentage of total motility for Kacang buck sperm post-thawing in this study was slightly higher than the findings reported by Morrell et al. (2022), who recorded a total motility rate of 59 ± 3%.

This study positioned semen in an Eppendorf tube 4 cm above liquid nitrogen. The results align with Kabir et al. (2024), who found that the optimal distance for the highest total motile buffalo sperm count (62.67±1.12%) is 1.6 inches, compared to 1.5 inches (47.13±3.37%) and 2 inches (38.10±1.97%). Ramirez-Reveco et al. (2016) found no significant effect of storage duration at -196°C on the percentage of total motile sperm after thawing, with values ranging from 60 ± 2.4% to 66 ± 3.2%. In contrast, Zamfirescu and Nadolu (2013) reported an approximately 50% reduction in total motility of alpine buck sperm, decreasing from 86.81% in fresh semen to 46.35% in frozen-thawed semen. The total post-thaw motility results in this study were higher than those reported for sheep, which showed a motility of 40.3 ± 1.7% (Marco-Jiménez et al., 2005). The total movement of sperm is the key for successful reproduction, and sperm with low motility is the primary cause of infertility in most cases (Aitken, 1995).

Packaging, temperature, and thawing duration significantly influence total sperm motility. In this study, semen was packaged in Eppendorf tubes and thawed at 50°C for 37 seconds, resulting in total sperm motility similar to some previous research. Chaiprasat et al. (2006) observed that higher thawing temperatures led to increased bovine sperm motility, with percentages of 51.04%, 40.41%, and 39.79% at thawing temperatures of 37°C, 30°C, and 25°C for 30 seconds, respectively. Samsudewa and Suryawijaya (2008) reported that thawing with ice water for 30 and 60 minutes resulted in 37.50±1.8% and 25.00±1.82% motility in Simmental cattle, and 40.00±1.64% and 20.00±1.14% in Limousin cattle. Straw water jacket packaging preserved sperm motility at a rate of 52% for up to 96 hours when stored in the refrigerator (Roca et al. 2004). Total sperm motility of 39±2.0 in the Tris-egg yolk extender, significantly higher than the 34±2.0, 33±4.0, and 33±4.0 observed in Tris-albumin extenders with 4%, and 6% (v/v) fig fruit filtrate, respectively, after four days of refrigeration (Triyani et al., 2024).

Progressive motility of sperm: Progressively motile sperm is a crucial parameter for assessing sperm quality after thawing (Abdelnour et al., 2020). The decrease in the percentage of progressive motility corresponds to the reduction in total motility (Table 2). Significantly higher (P < 0.05) percentages of progressive motility were observed immediately post-thawing and at 120 minutes post-thawing at T3 (39.13±1.02) compared to T1 (12.03±3.16) and T2 (21.21±1.26). This difference may be due to the use of Eppendorf tubes for frozen semen packaging, which are thicker than straw packaging, requiring a longer pre-freezing duration to achieve optimal quality of frozen sperm. Kabir et al. (2024) found that manually pre-freezing buffalo semen in minitube packages at 1.5 inches and 1.6 inches above the LN2 surface yielded sperm progressive motility of 24.50 ± 1.29% and 38.97 ± 1.10%, respectively. The progressive motility percentage of frozen semen post-thawing at T1 indicates its suitability for immediate consecutive insemination. For T2 and T3 (as shown in Table 2), the semen remains suitable for AI for up to 60 and 90 minutes post-thawing, respectively, according to the Standard Nasional Indonesia (2014).

 

Table 2: The impact of pre-freezing duration and room temperature storage time on the progressive motility of post-thaw kacang buck sperm packed in eppendorf tubes.

Post thawing (minute)	Treatments
	T1	T2	T3
0	49.31±1.21a	54.21±6.37b	56.19±2.05b
30	39.50±2.73a	47.23±1.05b	51.12±1.04c
60	26.12±1.03a	42.05±3.26b	46.11±1.11c
90	19.14±2.36a	31.18±1.11b	42.12±1.21c
120	12.03±3.16a	21.21±1.26b	39.13±1.02c

 

Note: Different superscripts in the same column indicate significant differences (P < 0.05).

 

Arifiantini and Yusuf (2006) found no significant differences in the percentages of progressively motile and live sperm when frozen in 0.3 ml minitub (52.16% and 69.4%) or 0.25 ml Cassou straw (49.59% and 70.44%). The post-thaw progressive motility percentages at T2 (54.21 ± 6.37) and T3 (56.19 ± 2.05) are consistent with Sitomorang’s (2002) findings of 56.0% and 51.5% for Tris-citrate extenders enriched with 1 mg/ml and 0.5 mg/ml cholesterol in straw packaging. White (1993) highlighted the low cholesterol levels in seminal plasma and sperm, making sperm vulnerable to cold shock. Therefore, the inclusion of 2.5% egg yolk (Evans and Maxwell, 1987) containing lipoproteins can help mitigate the effects of cold shock. Zaenuri and Rodiah, (2025) reported, after six hours of preservation at 27°C, spermatozoa in Tris-albumin extender with 8% (v/v) Fig fruit filtrate showed significantly higher progressive motility (66±2.53; P < 0.05) compared to the 4%, and 6% (59±4.1, 60±2.74, and 62±2.24, respectively).

Plasma membrane integrity: The integrity of the plasma membrane in sperm is a critical factor influencing their viability. An intact membrane correlates with prolonged viability, which in turn enhances both total motility and progressive motility of sperm. The integrity of the plasma membrane directly affects the quality of sperm (Pawar and Kaul, 2011). Physiologically, sperm membrane integrity plays a role in various vital processes that occur in sperm, including protecting and maintaining sperm motility in the female reproductive tract, capacitation and fertilization (Mocé and Graham, 2008). The proportion of high-quality sperm is influenced by the presence of sperm with normal membrane integrity (Makarevich et al., 2011).

 

Table 3: The impact of pre-freezing duration and room temperature storage time of eppendorf tube-packed semen on plasma membrane integrity parameters of post-thaw buck sperm.

Post thawing (minute)	Treatments
	T1	T2	T3
0	59.04±2.57a	64.04±1.07b	66.03±1.17b
30	41.09±1.27a	61.22±1.37b	63.09±2.16b
60	38.11±1.91a	46.17±2.05b	55.21±1.14c
90	31.14±1.08a	37.13±1.21b	47.17±3.06c
120	29.07±2.17a	33.04±2.17a	45.04±2.17c

 

Note: Different superscripts in the same column indicate significant differences (P < 0.05).

 

The percentage of sperm with intact plasma membrane (Table 3) at time point T1 was significantly lower (P < 0.05) compared to T2 and T3. Conversely, T3 showed a significantly higher percentage (P < 0.05) than T2 from 60 to 120 minutes post-thawing. Ramirez-Reveco et al. (2016) found that long-term storage at -196°C did not affect the plasma membrane integrity of bovine sperm, consistent with other studies. Morrell et al. (2022) reported 29.9 ± 14.7% membrane integrity in post-thawed buck sperm, while Barbas et al. (2024) observed a decline from 65.5% to 42.9% in buck sperm plasma membrane integrity pre- and post-thawing. Zaenuri et al. (2017) reported that the integrity of the plasma membrane of Kacang buck semen, when preserved in a Tris-egg yolk-based extender supplemented with 0.06 g (v/v) of free-drying fig fruit extract, was measured at 42.1 ± 5.1% after 92 h in refrigerated. Makarevich et al. (2011) found that plasma membrane integrity is crucial for sperm metabolism and function, making it necessary to recover a high percentage of the sperm with integrity of membrane after storage and processing.

The decrease in sperm integrity after thawing and room temperature storage may result from insufficient cholesterol in the extender. Arman (2014) noted that cholesterol is crucial for membrane integrity and sperm viability. Low cholesterol levels in semen and sperm increase cold shock susceptibility (White, 1993; Arman, 2014). Thus, more sperm with normal membranes leads to a higher percentage of high-quality sperm (Makarevich et al., 2011).

 

Table 4: The impact of pre-freezing duration and room temperature storage time on the viability of post-thaw buck sperm packaged in eppendorf tubes.

Post thawing (minutes)	Treatments
	T1	T2	T3
0	57.02±1.57a	65.01±0.07b	66.01±0.17b
30	53.02±1.17a	63.12±1.17b	65.02±1.12b
60	39.01±1.11a	47.07±1.05b	59.11±1.04c
90	34.11±2.08a	38.23±1.21a	54.12±1.05b
120	31.02±2.15a	30.08±1.17a	43.03±1.06c

 

Note: Different superscripts in the same column indicate significant differences (P < 0.05).

 

Viability of sperm: Sperm viability is also a critical factor in assessing sperm quality. The percentage of viable sperm at time point T1 was consistently significantly lower (P < 0.05) from the immediate post-thawing period up to 120 minutes thereafter, compared to time points T2 and T3 (Table 4). According to the sperm viability indicator, Kacang buck semen stored in Eppendorf tubes under T1 remained suitable for artificial insemination (AI) for up to 30 minutes post-thawing, while treatments T2 and T3 were suitable for 60 and 120 minutes post-thawing, respectively. Roca et al. (2004) found that sperm viability decreased along with preservation time. The sperm viability observed in this study was higher than the findings reported by Barbas et al. (2024), which indicated a decrease in viability from 75.0% before freezing to 42.8% post-thawing. The viability of post-thawed bull sperm, as reported by Putra and Ducha (2019), is 43.5 ± 1.5%.

The post-thaw viability percentage of buck sperm is reported to be 68±2.1, shows no significant decrease compared to samples stored for over ten years, with a viability of 60±1.8 (Ramirez-Reveco et al., 2016). In contrast, Malik et al. (2015) found that the viability and motility of bovine sperm are affected by the storage duration. Specifically, semen preserved at -196°C for six years showed lower viability and motility (64.50±0.39 and 42.08±0.13, respectively) than samples stored for one year (80.63±0.41 and 48.33±0.31, respectively). The equilibration rate of this study was 0.25 ºC/min, in line with Januskauskas et al. (1999) that no different effects of different cooling rates during equilibration of semen from room temperature to 4ᵒC, at 4.2ᵒC/min or at 0.1ᵒC/min on in vitro sperm viability, motility, membrane integrity, capacitation and acrosomal status post-thawing

It seems that enriched extender with antioxidant, energy sources or others effected to the sperm quality at cold or freezed. Putra and Ducha (2019) found that treating Boer buck semen samples with fructose alone or in combination with raffinose in a basic soya tris diluent resulted in the high post-thaw viability percentages, with values of 40.42 ± 0.44 and 41.77 ± 0.32, respectively. Arman (2014) noted that buck sperm can remain viable for extended periods, and fertility rates improve when cholesterol is added to the extender.

The utilization of Eppendorf tubes offers several advantages, including their accessibility, cost-effectiveness, and the capability of a single tube to facilitate the insemination of one to four female goats. However, it is important to emphasize that the fertilization potential of semen is not confirmed until the sample is used for insemination, regardless of the extender or packaging used.

CONCLUSIONS AND RECOMMENDATIONS

The manual, cost-effective semen freezing method in Eppendorf tube is suitable for small laboratories with limited facilities. Future research should explore how different extenders, equilibration, and pre-freezing periods affect post-thaw semen quality. Validation of fertilization rates compared to modern methods is essential.

ACKNOWLEDGEMENTS

The authors thank Professor Adji S. Dradjat for his valuable advice and Mr. Hananto, a technician in the Animal Reproduction and Genetics Laboratory at Mataram University for his assistance with semen processing and cryopreservation.

NOVELTY STATEMENTS

This study explored a manual, cost-effective semen freezing method using Eppendorf tubes and its effects on post-thaw Kacang buck semen quality, a previously underexplored area.

This method is ideal for small laboratories with limited resources.

AUTHOR’S CONTRIBUTIONS

All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

Conflict of Interest

The authors declare that they have no conflict of interests.

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