Gelatin zymography, MMP-2, MMP-9 Kangayam cattle, Bands.

Kangayam cattle are an excellent draught breed and are predominantly found in the Kangayam, Dharapuram, Perundurai, Karur, and Palani taluks of Tamil Nadu, southern India (Akila et al., 2012). According to the 19th Livestock Census, India has a cattle population of 190.9 million, making it a major livestock species. Cattle account for approximately 37.3% of India's total livestock population and 14.7% of the global cattle population. India has 43 registered native cattle breeds, broadly classified into dairy, draught, and dual-purpose breeds based on their primary utility in dairying or agricultural work. Indigenous farm animal breeds have adapted to harsh climatic conditions with minimal management inputs, including limited feed, fodder, and healthcare. Additionally, they efficiently convert low-quality feed into nutrient-rich outputs, making them well-suited to tropical environments and playing a crucial role in agriculture (Vinothraj et al., 2024).

Matrix metalloproteinases (MMPs) are a family of zinc-dependent enzymes involved in the degradation of extracellular matrix components. Among them, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) specifically target gelatin and type IV collagen, playing significant roles in various physiological processes in cattle. MMP-2 and MMP-9 are involved in the remodeling of the endometrial extracellular matrix during the estrous cycle and pregnancy. Their activity is regulated by various factors, including cytokines and hormones, ensuring proper tissue remodeling for successful implantation and maintenance of pregnancy.  The presence of MMP-2 and MMP-9 in bovine semen has been detected through gelatin zymography, suggesting their role in semen quality. These enzymes may influence sperm motility and overall fertility. 

Elevated levels of MMP-9 have been associated with inflammatory conditions such as mastitis in cattle. Monitoring MMP-9 activity can serve as a biomarker for early detection and management of such diseases. 

Assessing the activity of MMP-2 and MMP-9 in reproductive tissues and semen can provide insights into fertility status, assisting in the selection and breeding of high-quality livestock.  Hence, the present study was conducted to find out the presence of gelatinases in the Kangayam cattle breed of Tamil Nadu.

The current study was carried out at the Department of Veterinary Physiology and Biochemistry, TANUVAS-Veterinary College and Research Institute, Salem, Tamil Nadu, India.   

A. Collection and evaluation of serum

Twelve healthy male (Group I; n-12) and female animals (Group II; n-12) of the Kangayam breed of cattle were selected. Blood samples from each animal were collected in a heparinized vacutainer during the early morning before feeding the animals. The samples were transported to the laboratory immediately and evaluated for protein content using the standard procedure of Lowry’s method (Makowski and Ramsby 1996). The blood samples were centrifuged at 3000 rpm for 15 min, and the separated serum was analyzed for protein content by photometric estimation of blue color by using a spectrophotometer. The standard curve was built by using various concentrations of bovine serum albumin (BSA) as standard. The serum samples were stored at -20°C for further analysis.

B. Gelatin zymography

The serum samples were subjected to modified SDS-PAGE (modification of Laemmli’s method carried out by Heussen and Dowdle (1980) with the addition of a co-polymerizing substrate of gelatin (0.3%), and further procedures were carried out by the method described from our laboratory (Prakash Krupakaran et al., 2016).

C. Analyzing the results of gelatin zymography

Human capillary blood gelatinase was used as the standard marker for comparing the zymogram bands as per the procedure carried out by Makowski and Ramsby (1996), and further methods were described by Prakash, krupakaran et al. (2016).

Gelatin zymography was performed on all serum samples from the two groups, and the results are depicted in Fig. 1. Since every serum sample totally broke down the gelatin, they were all proteolytically active. Human capillary blood gelatinases were used as molecular weight markers, helping in the identification of MMP bands in the serum samples (Lane 1, 7, 13). Lane 2, 3, 10, 11, 12 (male cattle serum) and Lane 4, 5, 6, 8, 9 (female cattle serum). It was verified that the main bands were seen in both groups at 220, 135, and 92 kDa of MMP-9 and 72 kDa of MMP-2. The latent forms of MMP-9 and MMP-2 are represented by the two significant bands that were found in the Kangeyam breed at 92 kDa and 72 kDa, respectively. Additionally, two lytic bands representing the performs of MMP-9 were detected in each group at 220 and 135 kDa. However, the active forms of MMP-9 and MMP-2 are not found in any of the Kangeyam breed’s blood samples. Prakash Krupakaran et al. (2016) and Balamurugan et al. (2023) in bovine, Prakash Krupakaran et al. (2015) in bovine, and Balamurugan et al. (2017) in ovine species were all in agreement with the findings of the current study.

Effect of sex on the expression of gelatinase activity. To find out the relationship between the sexes, the gelatin zymograms of male and female animals were compared. In the Kangeyam breed, in male groups (G I: lane 2, 3, 10, 11, and 12), the intensity of the latent form of MMP-2 (72 kDa) was 1.5 times higher than that the female groups (G II: lanes: 4, 5, 6, 8, 9). In male groups (G I: lane 2, 3, 10, 11, and 12), two more prominent bands were noticed at 220, 125 kDa revealed that they are the lytic bands of MMP-9, indicating the pro-enzymatic forms of MMP-9. As compared to male groups, the female groups (G II: Lanes: 4, 5, 6, 8, 9) showed thicker bands at 125 and 220 kDa, indicating the expression of pro-enzymatic forms of MMP-9 was very clear. At 220 kDa, the samples from female animals showed more prominent bands than those of male animals. At 135 kDa of MMP-9 (pro MMP-9), no bands were observed in any of the samples, but at 125 kDa of MMP-9, all the samples showed prominent bands. In line with the present results, Prakash Krupakaran et al. (2016); Balamurugan et al. (2023) in bovine and Balamurugan et al. (2017) in ovine species were observed. But one difference observed in this study was that the preform of MMP-9 was observed at 125 kDa instead of 135 kDa. This might be due to breed variation and the individual physiological status of the animal.

The latent form of MMP-9 (92 kDa) was 1.5 times more intense in the female groups (G II: Lanes: 4, 5, 6, 8, 9) than in the male groups (G I lane 6, 8 and G II lane 7, 9). Similar observations were noticed in the earlier reports of Balamurugan et al. (2023) in bovine and Balamurugan et al. (2024) in buffaloes. In both male and female animals, the latent form of MMP-2 (72 kDa) was visible in all the animals. The intensity of MMP-2 (72 kDa) was 2-4 times higher than the latent form of MMP-9 (92 kDa). As compared to female group, the male group showed more intensity of MMP-2. This is corroborated with earlier reports of Balamurugan et al. (2023) in bovine, Balamurugan et al. (2024) in buffaloes, and Cancemi et al. 2020 in humans. According to a study by Cancemi et al. (2020), in humans, there was a discernible rise in Pro-MMP-2 activity with age in men but not in women. This is because, particularly in women, the expression of MMPs is also influenced by hormonal status (Berg et al., 2014). 

Fig. 1. Gelatin zymography of MMPs in serum of Kangayam cattle (13 microlitres of serum in each well).

The role of MMP-2 and MMP-9 could be further explored for their active role in various physiological mechanisms and how it augments the reproduction potential of the Kangeyam cattle. 

T.C. Balamurugan, V. Haripriya, R. Prakash Krupakaran, G. Anandhi, E.L. Aadhie Shrie, M. Vishal and M. Swathi  (2025). Gelatin Zymography of Matrix Metalloproteinases in Serum Samples of Kangayam Breed Cattle. Biological Forum, 17(2): 67-70.