Population Structure, Morphology, and Health Assessment of Philippine Swamp Buffalo (Bubalus kerabau, Fitzinger, 1860) in Calayan Island, Cagayan

Simple Summary

Information on the population of swamp buffalo in Calayan Island is necessary for useful conservation management programs within the country. The objective of this study was to better characterize the size, structure, and health statuses of these buffaloes. In comparison to other buffalo populations, Calayan swamp buffaloes are generally larger, except in body length, as indicated by the results. Using the optimized genetic markers, the study was able to identify the population structure and to differentiate between the swamp and river subspecies, as well as their crossbreds. The current study also included the first reports of surra and brucellosis as emerging diseases on the island. The results indicate the conservation and health-management potential of these buffaloes. It would be advisable to gather and conserve genetic materials from live specimens of the swamp buffalo population on Calayan Island.

Abstract

Understanding the state of the swamp buffalo population in Calayan Island is important to strengthen the conservation and management program in the country. This study aimed to provide insights into the morphology, population structure, and health profile of the swamp buffaloes on the island. In total, 35 fresh blood samples were analyzed using 27 polymorphic microsatellite markers to determine the population structure analysis. Data were gathered for the morphological features of Calayan swamp buffaloes and served as the baseline information for the descriptive traits. Furthermore, samples were tested for surra and brucellosis using PCR and serological tests, respectively. The results showed that Calayan swamp buffaloes were morphologically bigger, except for body length, compared with other populations. A genotype analysis using microsatellite markers showed remarkable discriminatory power to distinguish distinct populations within the tested population, and could discriminate subspecies of swamp and river types plus crossbreds. The study also reports the first incidence of surra and brucellosis on the island. Overall, the new insights provided on the newly detected Philippine carabao lineage in Calayan Island would be highly recommended for the ex situ conservation and animal health control strategy. The conservation strategy would encompass collecting, cryopreserving, and storing viable germplasms from local swamp buffalo on Calayan Island.

1. Introduction

Water buffalo (Bubalus kerabau, Fitzinger, 1860 swamp-type) is an economically important livestock species in Southeast Asia, including the Philippines. The Philippine carabao or native swamp buffalo was considered as the Philippines’ national animal. In addition, these species are robust and adaptable to harsh environments, and display resiliency or susceptibility to pathogens [1,2]. These characteristics play an essential role in areas where they thrive best. The native carabao is the farmer’s best companion as the source of draft power in rural farming communities. This animal was also the source of meat, milk, and organic fertilizer, which are important for a sustainable agriculture system [3]. Despite their economic contribution to the livelihood of the farmers and livestock industry, the Philippine carabao is facing threats and challenges.

In developing countries, the market demand for a consistent source of the product usually dictates the livestock industry trends. The domestic livestock sector’s response to the market signals has a negative tendency to reduce the number of breeds. In addition, others responded to market demand that required the utilization of imported genetic resources, which resulted in the loss of local or domestic animal diversity. The Food and Agriculture Organization (FAO) reported in 2000 on the status of the world’s breeds that had a loss of genetic variation: 12% have become extinct, 17% are endangered, 9% are in a critical condition, and 39% are not at risk [4]. In the Philippines, conserving swamp buffaloes would resolve various concerns such as uncontrolled crossbreeding, high animal market demand, and drastic effects due to climate change.

Through the artificial insemination (AI) service, crossbreeding is commonly practiced in the country to produce crossbred offspring (swamp × riverine) or upgraded swamp buffalo. The crossbreeding program aims to improve and increase milk and meat production [5]. The uncontrolled crossbreeding could result in the rapid loss of the native breeds in favor of the crossbred and riverine breeds. Since these improved breeds provide higher income to farmers, they would be easily enticed to exchange and sell their native animals. While they keep crossbreds and riverine breeds for milk and meat production, the high animal extraction rates favor the native carabao breed, since it can be easily sold and become a source of fast cash for the farmers.

The drastic effects of climate change could result in potential risks to animal health, primarily due to changes in environmental conditions [6]. For instance, livestock animals such as buffaloes suffer from nutrient deficiency, inadequate diet, and heat stress during extreme environments due to the non-availability of feed and poor pasture conditions [7]. The threats to sustaining the demands of local stocks are very alarming. Therefore, the appeal to go back to increase the production of the native buffalo breed, which could easily adapt to the effects of climate change, needs pressing attention.

In 2022, the Municipal Agriculture Office reported a total population of 7793 carabaos on Calayan Island. In addition, for several centuries on the island there was an absence of crossbreeding, introduction of riverine buffalo semen, or shipment of live bulls. This led the carabaos in the area to remain purebred and a strong candidate as the conservation area. Thus, this paper aimed to report on the morphometry and population structure to validate the breed identification, survey the animals’ health profile, and support concerted efforts by various stakeholders’ approaches for the strategic conservation and management of Philippine swamp buffalo on Calayan Island.

2. Materials and Methods

2.1. Sites and Sample Collection

This study was approved by the Department of Agriculture—Philippine Carabao Center Research Ethical Committee (BG-15002-RC), with Institutional Animal Care and Use Committee approval of handling animals (PCC-ACUP-005-2024). In addition, all experiments followed relevant local government unit (LGU) regulations and verbal consent from the local swamp buffalo farmers. A total of 79 unrelated swamp buffaloes were identified for this study (Supplementary Table S1). Due to the unavailability of pedigree records, the basis of measuring the relatedness of individual animals was performed through interviews with farmers to determine the absence of full siblings and half siblings among the animals before sampling collection. Whole-blood and serum samples were obtained in the jugular vein using a sterile vacutainer needle in EDTA-treated and plain vacutainer tubes from swamp buffaloes in Calayan Island, Cagayan. Additional unrelated swamp samples from Batanes, Benguet, and Ilocos Norte were included in the morphological and molecular analysis for population comparison (

Table 1. Distribution of samples in Northern Luzon provinces included in the study.

Species Name	No. of Sample	Location
Swamp buffalo	79	Calayan Island
Swamp buffalo	26	Batanes
Swamp buffalo	12	Benguet
Crossbred buffalo	16	Ilocos Norte
Riverine buffalo	5	Nueva Ecija

and Figure 1). The collected samples were stored in a cooler with coolants and transported to the laboratory for further investigation.

2.2. Morphological Characteristics and Physical Features

Morphological traits of swamp buffaloes and photo documentation for individual animals were recorded following the recommendation by Khan et al. [9]. Phenotypic characteristics, such as the body coat color, forehead, foreleg, hind leg, muzzle, and iris, were noted. In addition, the coat pattern, horn orientation, ear shape, and orientation were also recorded. The general body morphology, including the body length (BL), height at withers (HT), heart girth (HG), face length, and neck circumference, was also measured, and univariate analyses were performed on morphometric traits using a linear model with the effects of sex and age following the published linear model formula [10].

Y_jkl = μ + S_j + A_k + e_jkl

where

• Y_jkl = Individual observation;
• μ = Overall Mean;
• S_j = Effect of the jth sex;
• A_k = Effect of the kth age category of the buffalo;
• e_jkl = Random error.

2.3. DNA Isolation and PCR Optimization

The genomic DNA was extracted from the whole blood samples using the commercially available DNA extraction kit from RealiPrep™ Blood gDNA with some modifications including the additional centrifugation (14,000 rpm) for 1 min after the third washing to remove traces of cell wash solution (CWS) before the first elution and extension of 5 min incubation time of DNA in nuclease-free water before the elution step to obtain a high yield and good quality of DNA [11].

Twenty-seven polymorphic microsatellite markers recommended by the Food and Agriculture Organization (FAO) were amplified following the optimized PCR reactions and thermal cycler conditions [3]. The PCR products with various allele sizes were visualized using 2% agarose gel. Amplified PCR products were sent to 1stBASE Sequencing Malaysia for fragment analysis.

2.4. Microsatellite Analysis

The genotyping for each locus was scored using Geneious 10.1.3 software with an internal size standard of GeneScan™ 500LIZ™. Analysis of the genetic structure and membership coefficient (Q) was conducted using an alternative model-based Bayesian clustering analysis using STRUCTURE [12] and the optimum ∆K value was visualized and calculated using the STRUCTURE Harvester program [13].

2.5. Animal Health Screening

Seventy-nine whole blood samples were tested for Trypanosoma evansi using a PCR-based method targeting variable surface protein (RoTat 1.2 gene) according to a previous study [14]. Only 14 serum samples were tested for brucellosis using a serological rapid plates test (Bengatest^® Synbiotics, Kansas City, MI, USA) according to the manufacturer’s protocol, as the remaining samples were hemolyzed and unfit for testing.

3. Results

3.1. Morphological Characteristics and Physical Features

The morphological and physical features of swamp buffaloes in Calayan Island showed that the predominant coat color was black and gray, the muzzle and iris were black, and the stocking color of both forelegs and hindlegs was white (Figure 2). Moreover, all swamp buffaloes on the island showed a plain coat pattern. The average morphometric values of swamp buffaloes on the island with two other locations sampled in Northern Luzon are presented in

Table 2. Least squares estimate and descriptive morphometric traits of swamp buffaloes in Calayan Island, compared to Batanes and Benguet buffaloes.

Morphometric Traits	Calayan Island		Batanes		Benguet
	LSMean (SE)	Range (cm)	LSMean (SE)	Range (cm)	LSMean (SE)	Range (cm)
Body Length	114.0 (1.5)	62–147	137.4  (2.6)	112–164	125.8 (2.1)	102–144
Heart Girth	188.3  (1.5)	106–225	184.3 (2.8)	145–206	180.7 (2.3)	155–208
Height at withers	135.9  (1.2)	88–199	132.6 (2.2)	119–160	127.5 (1.8)	111–140
Neck circumference	97.2  (2.1)	77–123	96.4 (2.10)	73–149	95.4 (1.7)	66–120
Face Length	52.6  (0.8)	44–67	50.3 (0.78)	43–56	49.4 (0.6)	38–55
Horn Length
Horn greater curvature	59.5  (2.0)	37–81	54.4 (1.4)	22–82	49.5 (1.6)	23–68
Horn lesser curvature	47.0  (1.5)	29–64	43.9 (1.6)	20–70	38.9 (1.2)	14–53
Width between horns	16.5 (0.4)	14–20	18.0 (0.4)	14–24	19.4  (0.4)	12–23
Distance between horns	54.1 (3.0)	34–73	60.5  (3.0)	24–114	53.7 (2.4)	16–70

Highest LSMean (Bold fonts).

.

3.2. Population Structure

In total, 35 out of 79 animals were subjected to population structure analysis due to the elimination of full siblings and half siblings among the individuals to maximize the genetic diversity within the sampled population. The STRUCTURE analysis from K = 2 to K = 5 unveiled the presence of a population structure across swamp buffalo populations in the northern part of the Philippines (Figure 3). The K = 2 analysis revealed the clear separation of swamp and riverine buffaloes. At the optimum K = 3, the analysis showed the delineation of swamp buffaloes in Calayan Island and the other swamp buffalo populations in Northern Luzon that were included in the study. The swamp buffaloes in Calayan Island showed the highest average Q value of 0.998 as compared with Benguet (0.878), Batanes (0.852), and Ilocos (0.433). Additionally, incorporating riverine buffalo as a reference sample revealed that 43.7% of the Ilocos population (7/16) had male introgression of the riverine bloodline (

Table 3. Average membership coefficient of Calayan Island, Batanes, Benguet, Ilocos Norte, and riverine buffaloes at optimum ∆K = 3.

Collection Site	Average Q Value
	Cluster 1	Cluster 2	Cluster 3
Calayan Island	0.998	0.002	0.000
Batanes	0.852	0.137	0.011
Benguet	0.878	0.120	0.002
Ilocos Norte	0.433	0.120	0.447
Nueva Ecija	0.003	0.060	0.937

).

3.3. Animal Health Screening

A total of 79 samples was tested for surra and 14 samples for Brucella infection. The results showed a positive rate for surra and brucella spp. infection in the newly established native carabao sanctuary in Calayan Island of 2.53% (2/79) and 50% (7/14), respectively.

4. Discussion

The physical features of swamp buffaloes in Calayan showed that the coat color was predominantly black and gray. According to a study, black-colored animals were more likely heat intolerant, which was explained by their wallowing behavior [15]. On the other hand, the gray coat color of the animals on the island could be linked with previous phenotypic reports on the Calayan swamp buffaloes that they had whitish forelegs and hindlegs, which are among the diagnostic features of swamp buffaloes [10]. Moreover, in other Asian countries, such as in Bangladesh, there are also reports of gray coat color patterns in swamp buffaloes specifically found in the Sylhet district, Trishal, and Companiganj sub-district of Bangladesh [16,17].

In this study, the morphology data gathered provided the baseline information showing descriptive traits of the swamp buffaloes selected for conservation on the island. The average morphometric measurements of the buffaloes on Calayan Island revealed that they are generally tall and hefty but have a shorter body length compared to the swamp buffaloes in Benguet and Batanes Island. The body trait morphometry, specifically the height at the withers, heart girth, and body length, were the essential traits that were being considered for draft together with having sound feet, legs, and temperament. The heftiness can be extended to the neck circumference as well. The swamp buffalo head appeared longer but narrower on average, as indicated by the face length and the width between the horns. The horns were also generally longer but the distance between the horns, measured as the distance between the tips, was wider for buffaloes from Batanes Island, indicating the horns in Calayan may be more sickle-shaped or curved. Thus, the average measurements suggest that swamp buffaloes from Calayan appear to be bigger, except for in terms of body length.

The Bayesian analysis of the population structure of the Northern Luzon swamp buffalo populations indicated the presence of three clusters composed of Calayan Island (cluster 1), riverine buffaloes (cluster 2), and a genetic admixture of swamp buffaloes from Benguet, Batanes, and Ilocos Norte (cluster 3). Following the published Q value threshold of 0.80 in the Saler cattle breed [18], the increased Q value of 0.90 in all Calayan Island swamp buffaloes confirmed its distinctness from the rest of the Northern Luzon populations. According to studies, animals with an arbitrary cut-off of an 80% genetic membership coefficient were attributed as a pure population or specific cluster, while at below 80% they were considered admixture populations [18,19,20,21,22]. The presence of the distinct sub-populations reflects the old and recent population history of Philippine carabao, which is further explained by two factors. First, its isolation of population by distance. The geographic location of Calayan Island, situated about 24 miles west-south-west of Babuyan Island, Philippines, which requires approximately a six-hour boat ride, could explain why this island has a distinct population structure [10]. The island has not been connected to any land bridges in either Luzon or Taiwan since the Middle or Late Pleistocene [23]. The physical barrier provided by great bodies of water between Calayan Island and mainland Northern Luzon caused difficulty in inter-island transportation. This factor limits the island from agricultural opportunities for livelihood improvement offered by the national and local government programs. The second factor was the initiatives and efforts toward the Carabao sanctuary implemented by its LGU. In 2017, the Calayan LGU institutionalized a local ordinance that limits the introduction of riverine buffalo lineage to preserve its native carabao population. This initiative will further the establishment of the Carabao Sanctuaries on the island. The implementation of this ordinance was supported by the Department of Agriculture—Philippine Carabao Center, together with its regional center at Cagayan State University, and facilitated the construction of the Carabao facility on the island.

It was interesting to discover that some animals tested were positive for diseases that may harm livestock production. Surra, caused by Trypanosoma. evansii, is primarily transmitted by insect vectors, such as Tabanus spp., a biting fly [24,25], and blood-containing infectious trypanosomes are required for transmission from one animal to another. Surra was an exotic disease in the Philippines until the introduction of imported cavalry horses from China brought by the American soldiers during the American–Spanish war in the 19th century [26]. The first surra outbreak was recorded in 1901 in Manila and then spread to other provinces in Luzon Island, and resulted in high mortality in horses [24,26]. Moreover, brucellosis is transmitted through direct contact with the infected animal [27,28] and has zoonotic potential [28,29]. This finding is the first reported case of both diseases on Calayan Island. The island was isolated, and animals from other areas in the country were seldom introduced. Possible transmission of these diseases can occur by the movement of animals without being properly examined [26] before they travel to the island. The Calayan Island LGU, furthermore, has no program or disease surveillance among its livestock on the island. Therefore, the study reported the baseline information of the first incidence of swamp-positive in surra and brucellosis. This finding became the basis for strengthening the animal health program for swamp buffaloes on Calayan Island. The obtained knowledge also provided a pertinent document for the surra and brucellosis control program in swamp buffaloes.

5. Conclusions

In conclusion, the morphological and molecular characterizations of swamp buffaloes in Calayan Island are necessary for conservation and management. A comprehensive assessment of swamp buffaloes on Calayan Island revealed a population characterized by larger morphometric measurements and a distinct genetic profile, indicative of a purebred Philippine carabao lineage. Furthermore, this study reported the first incidence of surra and brucellosis on the island; thus an animal health control program in swamp buffaloes needs to be implemented. These findings highlight that the future of the Calayan swamp buffaloes requires efforts beyond the current undertaking. It is recommended to strengthen the in situ and expand the ex situ conservation and health management of the local swamp buffalo on the island.