Open Veterinary Journal, (2025), Vol. 15(9): 4548-4557

Research Article

10.5455/OVJ.2025.v15.i9.61

Prevalence and risk factors of repeat breeding in dairy cows in Cendana District, Enrekang Regency, South Sulawesi, Indonesia

Kalvin N. Pongkapadang¹, Amrin Amrin¹, Rian Hari Suharto¹, Muhammad Yusuf², Erick Vitus Gabriel Komba³, John King N. Layos⁴, Fika Yuliza Purba^1,5* and Subaedy Yusuf^5,6

¹Veterinary Medicine Study Program, Faculty of Medicine, Universitas Hasanuddin, Makassar, Indonesia

²Laboratory of Animal Reproduction, Department of Animal Production, Faculty of Animal Science, Universitas Hasanuddin, Makassar, Indonesia

³Department of Veterinary Medicine and Public Health, Sokoine University of Agriculture, Morogoro, Tanzania

⁴College of Agriculture and Forestry, Capiz State University, Mambusao, Philippines

⁵Center for Research and Development of Livestock Resources and Tropical Animals, Universitas Hasanuddin, Makassar, Indonesia

⁶Agricultural Science Doctoral Program, Postgraduate School, Universitas Hasanuddin, Makassar, Indonesia

*Corresponding Author: Fika Yuliza Purba. Veterinary Medicine Study Program, Faculty of Medicine, Universitas Hasanuddin, Makassar, Indonesia. Email: fikapurba [at] med.unhas.ac.id

Submitted: 15/01/2025 Revised: 15/07/2025 Accepted: 11/08/2025 Published: 30/09/2025

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Abstract

Background: Repeat breeding is a major reproductive disorder in dairy cows, resulting in extended calving intervals, increased insemination costs, and reduced milk production. In Indonesia, particularly among smallholder farms in Cendana District, Enrekang Regency, repeat breeding remains poorly studied despite its high prevalence and limited reproductive efficiency.

Aim: To examine the prevalence and risk factors of repeat breeding in dairy cows in Cendana District, Enrekang Regency, South Sulawesi, Indonesia.

Methods: This study used 163 pluriparous dairy cows with normal reproductive cycles and managed under a traditional system based on an intensive farming approach. The characteristic data of repeat breeding were studied at the individual and farmer levels. Data were collected from 48 farmers through a questionnaire and direct farm observation. The percentage of repeat breeding was analyzed descriptively, followed by a chi-square test and odds ratio to determine the association between the prevalence of repeat breeding and associated risk factors.

Results: The results showed that the percentage of repeat breeding at the cow and farmer levels was 28.8% and 60.4%, respectively. Despite the high proportion at the farmer level, the variables associated with knowledge and management practices were not related to the prevalence of repeat breeding. At the cow level, milk yield, herd size, and history of reproductive disorders were significantly related to the prevalence of repeat breeding. Cows producing less than 10 l of milk per day were 6.71 times more likely to experience repeat breeding than those producing 10–20 l of milk per day. Meanwhile, cows in herds with fewer than 10 cows had 3.57 times greater odds of repeat breeding than those in larger herds. Cows with a history of reproductive disorders had a 3.03 times higher chance of repeat breeding than those without a history of reproductive disorders.

Conclusion: Repeat breeding of dairy cows in Cendana District was prevalent at both the cow (28.8%) and farmer (60.4%) levels. Although no significant associations were found at the farmer level, the occurrence of repeat breeding was significantly associated with low milk yield, small herd size, and a history of reproductive disorders at the cow level. These findings highlight the importance of improving cow-level management practices to reduce the incidence of repeat breeding in smallholder dairy farms.

Keywords: Dairy cows, Enrekang regency, Repeat breeding, Risk factors.

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Introduction

Dairy cows are specific breeds that produce large quantities of milk and are widely considered essential for agricultural production. The consumption of milk and dairy products continues to increase rapidly worldwide, including in Indonesia, creating an attractive market for local producers (Priyanti and Soedjana, 2016). However, domestic milk production dropped significantly in 2023 due to the outbreak of Foot and Mouth Disease (Sahara et al., 2023). According to a recent US Department of Agriculture (USDA) Global Agricultural Information Network (GAIN) report, annual production has dropped by 35%–60%, leading to an imbalance between supply and demand (Darmawan and Osinski, 2024). This underlines the need for substantial imports of milk to meet consumer needs. Despite the challenges of milk supply and demand in Indonesia, the dairy farming sector is expected to rebound, fueled by favorable government initiatives that aim to stimulate market growth and investment opportunities. The combination of high demand and limited domestic production sets the stage for recovery, with experts predicting 17% growth in the Indonesian milk industry between 2025 and 2029 (CAGR percentage of dairy milk consumption).

Smallholder dairy farmers, who average three cows per farmer with less than 10 l of daily milk production per head, account for 77% of Indonesia’s fresh milk production (Daryanto et al., 2021). The use of inferior genetic breeds, poor feeding conditions, and poor farming practices can lead to low levels of efficiency and milk quality on small-scale dairy farms (Sutarno and Setyawan, 2015). Therefore, smallholder dairy farms need to be highly efficient and profitable to rebuild milk production in Indonesia. Identification of non-productive animals is also a priority to prevent economic losses.

In 2023, the population of Indonesian dairy cows reached 0.5 million, marking a 12.90% decrease from the previous year (Central Bureau of Statistics, 2023). Poor reproductive performance is a major problem in livestock, particularly in smallholder farms. Repeat breeding is one of the reproductive disorders among dairy cows. In this condition, cows show normal estrus signs at apparently normal intervals but repeatedly fail to become pregnant after at least three successive attempts, despite the absence of apparent anatomical abnormalities or infectious diseases (Zemjanis, 1980). Repeat breeding causes significant economic loss and has been extensively studied in various countries and areas. The previously reported prevalence of repeat breeding varied worldwide between 5% and 36% (Asaduzzaman et al., 2017; Deka et al., 2021), reaching a higher prevalence of 62% in Indonesia (Yusuf et al., 2012).

The physiological cause of repeat breeding is complex and multifactorial. It can be attributed to various causes, not only of individual cow origin but also related to defects or AI management (Gustafsson and Emanuelson, 2002; Saraswat and Purohit, 2016). Multiple factors, including nutritional deficiencies (Talukdar et al., 2016) and abnormal heat behavior or improper heat detection (Cummins et al., 2012; Sood et al., 2015), have been reported to contribute to the prevalence of repeat breeding. Other causes include mismanagement in AI (Walsh et al., 2011), endocrine dysfunctions (Sood et al., 2015), or subclinical endometritis (Salasel et al., 2010). Additionally, various risk factors, including genetic merit, age, parity, body condition, milk yield, peri- and postpartum imbalances, environmental conditions, management practices, production system, and number of cows per farm, can influence repeat breeding (Pérez-Marín and Quintela, 2023). This suggests that the prevalence of repeat breeding could be reduced and controlled.

Enrekang Regency is the center of milk production in the eastern part of Indonesia, with approximately 1,000 heads of dairy cows producing approximately 2,000 tons of milk in 2021 (Central Bureau of Statistics, 2022). Meanwhile, Cendana District has the highest population of dairy cows in Indonesia, with a total population of 462 heads, which is close to half of the dairy cows (Central Bureau of Statistics, 2022). As previously mentioned, the overall service per conception times, interval from calving to conception, duration of pregnancy, and calving interval of dairy cows in a smallholder farm in Enrekang regency were 2.05 ± 1.49 times, 214.79 ± 166.35 days, 274.51 ± 5.10 days, and 490.16 ± 167.81 days (data in Mean ± SD), respectively (Yusuf and Sahiruddin, 2020). The prevalence of repeat breeding in dairy cows in Enrekang regency was reported at 31.57% and 29.62% for primiparous and pluriparous, respectively (Muhuruna et al., 2023). However, the associated risk factors have not been evaluated. While previous research has assessed the prevalence of repeat breeding in Enrekang Regency using non-probability sampling with the purposive sampling method, this study specifically focuses on Cendana District through a census method and further explores significant risk factors contributing to repeat breeding. Therefore, this study aimed to examine the prevalence of repeat breeding in dairy cows in Cendana District and identify the associated factors at individual cow and farmer levels, based on data collected from farmers and direct observation on the farm.

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Materials and Methods

Time and location

This retrospective cross-sectional study was conducted in June 2023 in Enrekang Regency, South Sulawesi, Indonesia, at geographical coordinates 3°31’57”S, 119°53’9”E.

Study population and design

According to the national census, Enrekang Regency had 1,016 dairy cows in 2022, with the highest population in Cendana district, accounting for 462 heads (Central Bureau of Statistics, 2022). However, the population was restructured by direct observation and interviewing local animal health officers. Approximately 267 heads were identified and structured based on age and sex. Among the 267 heads, 45 were males, and 222 were females, classified as lactating cows (n=163) and heifers (n=59).

This study used primiparous and pluriparous cows (n=163) with normal estrous cycles and managed under the traditional intensive system. At the cow level, repeat breeding was defined as a condition in which a cow fails to conceive after being inseminated three times with fertile semen, despite showing normal estrous cycles and no apparent clinical abnormalities of the reproductive tract. Meanwhile, due to the smallholder nature of dairy farming in Indonesia, where most farmers own fewer than 10 cows, repeat breeding was also assessed at the farmer level. A farmer was considered to have repeat breeding if at least one of their cows met the repeat breeder criteria, consistent with approaches used in similar studies conducted in Indonesia (Prihatno et al., 2013; Arningdiah et al., 2024).

The data include the number of cows with repeat breeding and risk factors related to repeat breeding at the cow and farmer levels. A non-disclosure agreement about the farmers’ participation was secured before the study was conducted. All responses were treated with confidentiality, and participation was voluntary. This study was approved by the Universitas Hasanuddin Animal Research Committee (Approval No. 014/UN4.1.RSHUH/B/PP36/2025).

Study variables

Variables related to individual cows, including age, parity, milk yield, herd size, duration of estrous detection per day, timing of AI, history of reproductive disorders, and body condition score, were collected by interviewing the farmer and directly observing the farm. Meanwhile, variables related to farmer, including age, education, gender, number of cows owned, farming experience, knowledge of estrous cycle, knowledge of estrous signs, AI timing, distance from inseminator to the farm, barn floor type, barn hygiene, sewer condition, water source, feed, and water container availability, were collected from 48 farmers through a questionnaire.

Data analysis

Each variable of interest was stored in Microsoft Excel and descriptively analyzed. The prevalence of repeat breeding at the cow and farmer levels was described as the percentage of the disease in the population at risk. Furthermore, chi-square tests were used to assess associations between categorical variables and the occurrence of RB. For categorical variables with more than two groups that showed a statistically significant association with repeat breeding in the overall chi-square test, post hoc pairwise comparisons were performed using Fisher’s exact test. To assess the strength of association, odds ratios (ORs) and their 95% confidence intervals (CIs) were calculated for each pairwise comparison. To control for the increased risk of Type I error due to multiple testing, the Bonferroni correction was applied to adjust the p-values.

Ethical approval

Not needed for this study.

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Results

Table 1 presents the summary data of repeat breeding prevalence at both the cow and farmer levels. This study collected both cow- and farmer-level data to assess the prevalence of repeat breeding and identify associated risk factors. At the cow level, data were obtained from 163 pluriparous dairy cows, including age, parity, milk yield, herd size, estrous detection duration, AI timing, history of reproductive disorders, and body condition score (Table 2). At the farmer level, responses from 48 dairy farmers were recorded, covering demographic characteristics (age, gender, education, and occupation) and management-related variables, including farming experience, number of cows owned, knowledge of estrous cycle and signs, timing of AI, distance from inseminator, barn conditions (floor type, hygiene, and sewer system), water source, and feed and water container availability (Table 3). The following subsections present detailed results on farmers’ sociodemographic profile, the prevalence of repeat breeding, and analysis of its associated risk factors.

Table 1. Occurrence of repeat breeding at the individual cow and farmer levels.

Table 2. Analysis of risk factors for repeat breeding at the cows level in Cendana district, Enrekang regency, South Sulawesi.

Table 3. Analysis of risk factors for repeat breeding at the farmer level in Cendana District, Enrekang Regency, South Sulawesi.

Sociodemographic characteristics

The sociodemographic characteristics showed that most farmers were 40 years old or older and were dominated by males, as described in Table 4. More than half of the farmers had a high degree of education, although most only worked for a living.

Table 4. Sociodemographic characteristics of farmer respondents (n=48) in Cendana district, Enrekang regency, South Sulawesi, Indonesia.

Prevalence of repeat breeding

The prevalence of repeat breeding was 28.8% (47/163) and 60.4% (29/48) at the individual and farmer levels, respectively (Table 1).

Analysis of risk factors at the cow level

At the cow level, where repeat breeding was detected in 28.8%, several risk factors were analyzed to determine the associated factors (Table 2). No significant association (p > 0.05) was observed between factors such as age, parity, timing of AI, and body condition score with repeat breeding. However, milk yield, herd size, and history of reproductive disorders were significantly associated (p < 0.05). Among cows producing less than 10 l of milk per day, 54.3% were repeat breeders, compared with only 15.1% among cows producing 10–20 l of milk per day. Although the overall chi-square test for milk yield was significant (χ²=27.888, p < 0.001), the group producing >20 l/day had no cows and was excluded from the post-hoc analysis. Cows producing less than 10 l/day had 6.71 times the odds of repeat breeding compared with those producing 10–20 l/day.

For herd size, repeat breeding occurred in 36.8% of cows in herds with fewer than 10 cows, compared with 8.6% in herds of 10–20 cows and 5% in herds of more than 20 cows. A significant association was found between herd size and repeat breeding (χ²=10.674, p=0.005). Cows in herds with fewer than 10 cows had 3.57 times greater odds of repeat breeding than those in larger herds. Post hoc pairwise comparisons were performed between the three herd size categories (<10, 10–20, and >20 heads), and p-values were adjusted using Bonferroni correction to account for multiple comparisons (Supplementary File). The comparison between cows in herds with fewer than 10 animals and those in herds with more than 20 animals revealed a statistically significant difference, with cows in smaller herds having 3.49 times higher odds of experiencing repeat breeding (OR=3.49, 95% CI: 1.25–9.74; Bonferroni-adjusted p=0.0370). Although the comparison between <10 and 10–20 head herds suggested elevated odds (OR=3.69, 95% CI: 1.02–13.26), it did not remain significant after adjustment for multiple comparisons (Bonferroni-adjusted p=0.1357). No significant difference was observed between the 10–20 and >20 head herd groups (p=1.000).

Finally, 68.4% of the cows with a history of reproductive disorders were repeat breeders, whereas only 23.6% of the cows without such a history experienced repeat breeding. A significant association was found between a history of reproductive disorders and repeat breeding (χ²=8.639, p=0.003). Cows with a history of reproductive disorders had a 3.03 times higher chance of repeat breeding than those without a history of reproductive disorders.

Analysis of risk factors at the farmer level

Despite the high prevalence of repeat breeding at the farmer level (60.4%), no significant relationships (p > 0.05) were found between the high prevalence of repeat breeding and any variables investigated due to a lack of response variance (Table 3).

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Discussion

Enrekang Regency has the largest dairy population in the eastern part of Indonesia. However, the Central Bureau of Statistics showed that the population of dairy cows has decreased considerably since 2020, at 1011, 1026, 1016, and 565 heads in 2021, 2022, and 565 heads in 2023 (Central Bureau of Statistics, 2024). This might be due to the typically low reproductive performance of smallholder farms. Yusuf and Sahiruddin (2020) stated that the reproductive performance of dairy cows in Enrekang Regency was 2.05 ± 1.49 times, 214.79 ± 166.35 days, 274.51 ± 5.10 days, and 490.16 ± 167.81 days for service per conception times, interval from calving to conception, duration of pregnancy, and calving interval, respectively. Furthermore, the prevalence of repeat breeding in dairy cows reached 31.57% for primiparous and 29.62% for pluriparous cows in 2021 (Muhuruna et al., 2023).

According to the sociodemographic results, which were similar to Nadhira and Sumarti (2017), most farmers were men because women often do not fully control livestock farming in Indonesia, and decision-making by females remains primarily at the household level. The current problem in the agriculture sector is the low youth interest in becoming farmers, with only 4.2% of farmers being old. This condition may threaten the sustainability of future agricultural businesses (Prayoga et al., 2020; Nurlaela et al., 2022). However, in addition to age, education level may influence access to information and understanding of management practices (Athambawa et al., 2021).

Owing to the importance of the farm economy, repeat breeding has been intensively investigated in many countries and areas. Juliana et al. (2015) reported a prevalence of repeat breeding in beef Bali cows in Pringsewu Regency of 19.85%, while Nasution et al. (2021) reported a prevalence of 17.3% in beef cattle in North Labuhanbatu Regency. Similarly, Subagio et al. (2019) found a 21.12% prevalence of repeat breeding in Jombang beef cows. Yusuf et al. (2012) and Damayanti et al. (2020) also reported a higher incidence of repeat breeding in dairy cows in Sinjai and beef cattle in Banyuwangi Regency, reaching 62% and 64%, respectively. At the farmer level, the prevalence rate of repeat breeding among dairy cow farmers was 20.64% in Batu and 29.4% in Daerah Istimewa Yogyakarta (Prihatno et al., 2013; Arningdiah et al., 2024).

The results showed that the prevalence of repeat breeding in Cendana District, Enrekang Regency, at the cow level (28.8%) was comparable to recent studies by Muhuruna et al. (2023), which observed a 31.57% and 29.62% prevalence for primiparous and pluriparous cows, respectively. In contrast, the prevalence of repeat breeding at the farmer level (60.4%) was significantly higher than that in previous studies. The prevalence of repeat breeding may differ based on the characteristics of the area and sociodemographics. In Indonesian smallholder farms, where livestock are traditionally managed without culling or replacement management, the prevalence of repeat breeding remains high, which is a major source of concern.

Based on the results, 65% (106/163) of the dairy cows provided milk yields ranging from 10 to 20 l. This is still less than the blueprint made by the Indonesian Director General of Livestock and Animal Health, stating that dairy cow productivity must reach more than 20 l per day. However, the result is in line with the current average milk production of Indonesian dairy cows, which ranges from 10 to 12 l per day (Pramono et al., 2023). Cows producing less than 10 l of milk per day were 6.71 times more likely to experience repeat breeding than those producing 10–20 l of milk per day. This result differs from that of Eshete et al. (2023), who reported that lower fertility was associated with higher milk supply. A commonly held theory is that high milk production and the metabolic environment supporting high milk production lead to the dysfunction of the innate immune system postpartum, leading to a failure of fertility (Berry et al., 2016). Furthermore, several studies have found a negative relationship between milk yield and fertility (Lucy, 2001; López-Gatius et al., 2006; Cutullic et al., 2012; Eshete et al., 2023).

While high milk yield has been associated with decreased fertility in intensive dairy production systems (Lucy et al., 2001), our findings suggest that, in the context of smallholder dairy farms in Indonesia, cows with lower milk yield (<10 l/day) exhibited a higher prevalence of repeat breeding. This observation may not contradict the global consensus but rather reflects a different production and management context, where low productivity, rather than genetic efficiency, may indicate suboptimal health, poor nutrition, or reproductive challenges. Nonetheless, we acknowledge that our data did not show a statistically significant association between BCS and repeat breeding, and further research is needed to explore the underlying mechanisms linking low yield and fertility outcomes in smallholder systems.

Dairy cow management in Enrekang Regency has traditionally been intensive, with each herd containing fewer than 10 heads. Herd size was significantly associated with repeat breeding in this study. Herds with fewer than 10 cows had a 3.57 times higher chance of repeat breeding than those with more than 10 cows. The density of livestock in a pen can affect the physiological condition and livestock management. This result differs from those of previous studies, which stated that the prevalence and incidence of repeat breeding significantly increased progressively from small to large herd-size dairy farms (Eshete et al., 2023). Additionally, farms with fewer dairy cows showed superior heat detection than those with more dairy cows (Asaduzzaman et al., 2017). Despite the considerable association between herd size and the prevalence of repeat breeding, it may not be related to density or physiological stress in animals.

The results showed that over half of the cow population (100/163) was in productive years, aged 4–6 years, but only 44.8% (73/163) had 1 or 2 offspring(s). This suggests that dairy cows in Cendana District have low reproductive performance despite the widespread use of reproductive technology such as AI. Some reproductive disorders, including retained fetal membranes, dystocia, endometritis, and mastitis, were also detected in 11.7% (19/163) of dairy cows. Furthermore, a history of reproductive disorders was significantly associated with the prevalence of repeat breeding. About 68.4% of cows with a history of reproductive disorders were determined as repeat breeders. Cows with a history of reproductive disorders had a 3.03 times higher chance of repeat breeding than normal cows.

Peripartum diseases and an increase in parity are associated with inferior reproductive performance, which increases the probability of cows being classified as repeat breeders, although repeat breeding syndrome is not solely associated with peripartum diseases (Bonneville-Hébert et al., 2011). The data also showed that among 19 cases, the reproductive problems cited were retained fetal membranes (10.5%, n=2), dystocia (36.8%, n=7), endometritis (36.8%, n=7), and mastitis (15.8%, n=3). Cows with a history of endometritis, mastitis, or retained fetal membranes were all classified as repeat breeding, but only one out of seven had a history of dystocia. In line with a previous study, cows with a history of reproductive issues, including abortion, dystocia, retained placenta, metritis/endometritis, and vaginal/uterine prolapses during the previous calving had a considerably greater prevalence of repeat breeding (Salasel et al., 2010; Eshete et al., 2023).

Cendana District has the largest dairy cow population in Enrekang Regency, with approximately 48 farmers. Livestock farming is dominated by older adult farmers (>40 years) with >10 years of experience. Management was often implemented through an AI-based intensive farming system. Most farmers followed standard dairy cow management practices and acquired regular training from local veterinary authorities. The majority were also knowledgeable about estrous and the estrous cycle, as well as how to maintain appropriate farming management and practices, including hygiene, sewer, feed, and water availability. No significant association was found between repeat breeding and risk factors at the farmer level. However, education level (p=0.098) and farming experience (p=0.068) were closely related.

This study contributes novel insights by identifying and analyzing specific risk factors associated with repeat breeding in smallholder dairy cows—an area that has been underexplored in previous research. While earlier studies in Enrekang Regency have reported the prevalence and reproductive performance of repeat breeders, the underlying determinants contributing to the condition were not investigated. This study provides new and region-specific evidence that low milk yield, small herd size, and a history of reproductive disorders are significantly associated with repeat breeding by employing a census approach focused on Cendana District and including both cow- and farmer-level variables. These findings are particularly relevant to Indonesia’s smallholder dairy systems and may inform targeted interventions to improve reproductive performance in similar contexts.

However, this study had certain limitations that could lead to biases. First, the area explored may not be representative of the entire dairy cow population in Enrekang Regency. Second, cows were identified as repeat breeding based on secondary data and information provided by local animal health officers and farmers. Third, some farmers were unwilling to provide specific information on current cow conditions, making data collection difficult. Fourth, all cows in this study were Friesian Holstein crossbreeds; however, specific data on days in milk, exact lactation stage, and complete parity distribution were not uniformly recorded during data collection, which limits the ability to control for these variables in the analysis of milk yield and reproductive outcomes.

In conclusion, the prevalence of repeat breeding in dairy cows in Cendana District, Enrekang Regency, was 28.8% at the individual cow level and 60.4% at the farmer level, indicating a substantial reproductive challenge in this smallholder farming system. Among the cow-level factors, low milk yield (<10 l/day), small herd size (<10 cows), and a history of reproductive disorders were significantly associated with an increased likelihood of repeat breeding. Although these findings contrast with studies from high-producing dairy systems—where higher milk yield is typically linked to poorer fertility—the results reflect the unique conditions of smallholder farms in Indonesia, where low productivity may be indicative of poor nutritional status or chronic health issues. No significant associations were observed at the farmer level, possibly due to the limited variation in the management practices of the respondents. These results indicate that repeat breeding in smallholder dairy cows is a multifactorial problem primarily influenced by intrinsic animal factors. Therefore, targeted interventions focusing on nutritional improvement, reproductive health monitoring, and herd management strategies are essential to reduce the prevalence of repeat breeding and improve reproductive performance in these systems.

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Acknowledgments

The authors are grateful to the farmers in Cendana District, Enrekang Regency, South Sulawesi, for their gracious cooperation during data collection.

Conflict of interest

The authors have no conflicts of interest to declare.

Funding

This study was supported and funded by the Faculty of Medicine, Universitas Hasanuddin through the Decree of the Dean of Faculty of Medicine No. 04486/UN4.6/KEP/2024.

Author’s contribution

FYP designed the study. KNP, A, and FYP conducted the study, analyzed the data, and wrote the manuscript. FYP, RHS, and MY supervised the study. FYP, RSH, MY, EVGK, and JKNL reviewed and revised the manuscript.

Data availability

All data supporting the results are available in the revised manuscript.

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NaN=insufficient data or zero counts in one or more categories, preventing chi-square calculation; NS=non-significant; S=significant. NaN=insufficient data or zero counts in one or more categories, preventing chi-square calculation; NS=non-significant; S=significant.