Effect of Giloy (Tinospora cordifolia) on seminal parameters of boar spermatozoa during preservation

Introduction

Livestock husbandry plays a pivotal role in global agriculture by contributing substantially to food security, rural livelihoods and economic development. Among livestock species, the pig (Sus scrofa domesticus) is considered one of the most eficient meat-producing animals due to its rapid growth, high reproductive potential and eficient feed conversion ratio (Thomas et al., 2025) [1] . Pork is an important source of high-quality animal protein worldwide, and the expanding swine industry requires continuous genetic improvement and optimized reproductive management to achieve sustainable production (Thomas et al., 2021) [2] . Artificial insemination (AI) is a major reproductive biotechnology extensively employed in swine production for accelerating genetic gain through the dissemination of superior germplasm from genetically elite boars to a large number of females. It ofers advantages over natural mating, including eficient utilization of superior sires, improved reproductive management and reduced risk of disease transmission (Foote, 2002; Lopez et al., 2017) [3,4] . However, the success of AI largely depends on the maintenance of sperm quality during semen preservation. Preservationinduced oxidative stress, resulting from excessive generation of reactive oxygen species (ROS) relative to antioxidant defense capacity, can adversely afect sperm membrane integrity, mitochondrial function, motility and fertilizing competence. Although physiological concentrations of ROS are involved in capacitation, hyperactivation and the acrosome reaction, excessive ROS induces lipid peroxidation and oxidative damage to proteins and nucleic acids (Aitken and Clarkson, 1987). Giloy (Tinospora cordifolia), a medicinal plant of the family Menispermaceae, contains diverse bioactive phytoconstituents, including alkaloids, diterpenoid lactones, steroids, glycosides, phenolics and polysaccharides, which possess documented antioxidant, anti inflammatory, immunomodulatory and cytoprotective properties (Saha and Ghosh, 2012; Gupta et al., 2024) [5,6] . Its antioxidant potential may therefore provide protection against preservationassociated oxidative injury in spermatozoa. However, the potential of T. cordifolia as a natural antioxidant supplement for maintaining boar semen quality and enzymatic homeostasis during preservation remains inadequately explored. Therefore, the present study was undertaken to evaluate the efect of Giloy (T. cordifolia) supplementation on the enzymatic profile and semen quality characteristics of boar spermatozoa during preservation, with particular emphasis on its potential protective role against preservationinduced oxidative stress.

Materials and Methods

Location and Experimental Animals

The study was conducted in the Department of Animal Reproduction, Gynaecology and Obstetrics, College of Veterinary Science and Animal Husbandry, Rewa, NDVSU, Jabalpur, Madhya Pradesh. Healthy breeding boars maintained at the Instructional Pig Unit of the Livestock Farm Complex were utilized.

Semen Collection and Experimental Design

A total of 12 ejaculates were collected using the double-gloved hand technique with a dummy. The gel fraction was removed using a gauze-filtered semen collection funnel. Ejaculates were transported to the laboratory at approximately 37 °C. Only samples exhibiting ≥70% progressive motility and sperm concentration >100×10 ⁶ spermatozoa/ml were included. Each ejaculate was divided into four equal aliquots. Group I served as control and received BTS extender without Giloy (Tinospora cordifolia) extract. Groups II, III and IV were supplemented with T. cordifolia extract at 100, 300 and 500 μg / 25×10 ⁶ spermatozoa, respectively. Following equilibration at 22 °C for 2 h, semen was diluted with BTS to a final concentration of 30×10 ⁶ spermatozoa/ml and preserved at 15% for 72 h. Assessments were performed at 24, 48 and 72 h of preservation.

Preparation of Giloy Extract

Authenticated T. cordifolia stem material was dried at 40 °C, powdered and subjected to Soxhlet extraction using 70% ethanol. The solvent was removed under reduced pressure using a rotary evaporator, and the concentrated extract was stored at 4 °C until use.

Semen Quality Evaluation

Semen samples were evaluated for progressive motility, sperm concentration, viability, morphological abnormalities, acrosomal integrity and functional plasma membrane integrity using CASA, eosin-nigrosin staining, Giemsa staining and hypo-osmotic swelling test (HOST), respectively.

Biochemical and Antioxidant Assessment

Seminal plasma was analyzed for catalase (CAT), superoxide dismutase (SOD), glutamate oxaloacetate transaminase (GOT), lactate dehydrogenase (LDH), lipid peroxidation (MDA) and total antioxidant capacity (TAC) using established spectrophotometric and ELISA-based methods.

Statistical Analysis

Data were analyzed using two-way analysis of variance (ANOVA) in IBM SPSS Statistics version 22.0 to determine the efects of treatment, preservation period and their interaction. Statistical procedures were performed according to Snedecor and Cochran (1994) [7] .

Results and Discussion

The present research work entitled "the efect of Giloy (Tinospora cordifolia) on seminal parameters of boar spermatozoa during preservation" was carried out in the Department of Veterinary Gynaecology and Obstetrics, College of Veterinary Science and Animal Husbandry, Rewa (NDVSU, Jabalpur), Madhya Pradesh. In the present study, breeding boar having age between 1.0 to 2.0 years and weighing around 80-100 kg were used for the collection of semen. Semen was collected twice a week by double glove hand method and was evaluated for the various seminal atributes e.g. volume (ml), seminal pH, colour, consistency, motility, sperm concentration (millions/ml). The extended semen was evaluated at diferent groups of preservation for initial progressive motility, spermatozoa viability and abnormality, acrosomal integrity, HOST, enzymatic profiles (LDH, GOT, Catalase, SOD), LPO and TAC. Results obtained in the study are writen under following sub headings:

Physico-morphological evaluation of fresh boar semen

The physico-morphological profiles of boar ejaculates were evaluated immediately postcollection to confirm physiological normalcy. The mean values for the key physicomorphological parameters are detailed in Table 2.0. [8] and Saini et al. (2016) [9] .

Spermatozoal functional microscopic characteristics 4.2.1. Initial progressive motility of boar spermatozoa

The initial progressive motility (%) at 0, 24, 48 and 72 hours in control (Group 1) as well as Giloy (Tinospora cordifolia) treated groups (Group 2, 3 and 4) were analyzed and results indicated that at 0 hours, initial progressive motility (%) did not difer significantly (p > 0.05) among the experimental groups, maintaining a baseline range of 85.57±1.91 to 86.65±2.96 as detailed in Table 3.0 and Figure 1.0. Temporal preservation induced a significant, linear decline (p < 0.05) in initial progressive motility across all groups from 24 to 72 hours.

Morphological abnormalities

The incidence of morphological abnormalities (%) in boar spermatozoa was observed and results indicate that in boar spermatozoa morphological abnormalities increased over time across all preservation setups (p < 0.05, Table 5.0 and Figure 3.0). At 0 hours, aberrant morphology remained uniform across groups, ranging from 11.89±0.29% to 12.47±0.29%. A, B, C, D) androw (a, b, c) with diferent superscripts difer significantly (p < 0.05).

During intermediate storage phases (24 and 48 hours), Giloy (Tinospora cordifolia) extract supplementation in Group 2 and Group 3 significantly suppressed (p < 0.05) the rate of morphological degradation compared to the control group. However, by 72 hours of preservation, the protective threshold was surpassed, resulting in statistically equivalent structural abnormality rates (p>0.05) across all four treatment groups, maximizing between 14.83±0.53% to 15.83±0.24%. In the present study, the percent of abnormal spermatozoa was ranging from 10.83±0.30 to 15.50±0.50 and this was in accordance with the findings of Chaudhary et al., 2017 and Singh et al., (2020).

Acrosomal Integrity

The maintenance of an intact acrosome is crucial for fertilization and these metrics was observed and results are summarized in the Table 6.0 and Figure 4.0. No baseline disparities were observed at hours (p>0.05). Storage-induced stress caused significant time-dependent acrosomal degradation (p < 0.05) in all groups.At 24 and 48 hours, all Giloy (Tinospora cordifolia)-treated groups (Group 2, 3 and 4) exhibited significantly higher acrosomal stability (p < 0.05) compared to control i.e. Group 1. By 72 hours, Group 2 and 3 displayed the highest acrosomal retention (64.58±2.87% and 64.83±2.12%, respectively), which was significantly greater (p < 0.05) than Group 4 (62.03±1.93%) and the control group (52.83±2.92%).

Hypo-Osmotic Swelling Test (HOST) responsiveness

The functional integrity of the tail plasma membrane was assessed via HOST and results are shown in the Table 7 and Figure 5. Initial responsiveness was statistically uniform (p>0.05) across the groups. Extended preservation caused a notable decline (p < 0.05) in membrane responsiveness over 72 hours. A, B, C, D) androw (a, b, c) with diferent superscripts difer significantly (p < 0.05).

Throughout the temporal evaluation (24, 48 and 72 hours), Group 3 consistently yielded significantly higher HOSTpositive percentages (p < 0.05) than the remaining groups. At 72 hours, membrane responsiveness was optimally preserved in Group 3 (62.92±2.47%), followed by Groups 4 (60.23±2.31%) and 2 (59.92±1.53%), while the control group reached a minimum value of 51.04±0.74% The percent HOST positive sperm in preserved semen reported as 60.56±2.93 in crossbred boar (Singh et al., 2017) [10] ; 56.83±0.90 in boar (Wafa et al., 2021).

Seminal Enzymatic and Oxidative Biomarkers 4.3.1. Catalase and Superoxide Dismutase an endogenous antioxidant enzyme kinetics

The activities of enzyme Catalase and Superoxide Dismutase (SOD) were observed and results are exhibited in the Tables 8.0 and Figures 6.0 and 7.0, respectively. For both enzymes, Group 1 (control) demonstrated a significant, time-dependent surge (p < 0.05) in raw activity levels, peaking at 72 hours (Catalase: 3.62±0.04 U/ml; SOD: 5.11±0.14U/ml). A, B, C, D) androw (a, b, c) with diferent superscripts difer significantly (p < 0.05) Conversely, the addition of Giloy (Tinospora cordifolia) extract successfully checked this compensatory enzyme up regulation. In Groups 2, 3 and 4, both Catalase and SOD activities remained suppressed and statistically equivalent (p>0.05) across the 24-, 48-and 72-hour intervals. This indicates a reduced requirement for endogenous enzyme synthesis due to the exogenous antioxidant protection provided by the Giloy extract. In our study, the SOD enzyme activity of spermatozoa at a fresh and preserved stage was ranging from 29.64±0.48 to 22.63±0.42. Our findings are accordance with the findings of Kadirvel et al., (2014). In our study the catalase activity of spermatozoa at a preserved semen was ranging from 0.77±0.10 to 0.41±0.11. It was seen that the lower finding is found with accordance Gebreselassie, 2009 (1.10±0.09) ; and higher finding are seen with the accordance Lone et aI., 2018 (0.001±0.00nM / mg)

Glutamic-Oxaloacetic transaminase and Lactate Dehydrogenase enzyme activity

The activity of Glutamic-Oxaloacetic Transaminase (GOT) and Lactate Dehydrogenase (LDH) into the extracellular medium was observed and are depicted in the Tables 10.0 and 11.0, Figure 8.0 and 9.0, respectively. Extracellular GOT levels rose significantly over time in the control group (p < 0.05) reaching a peak 1258.49±9.59 U/L at 72 hours. Groups treated with Giloy (Tinospora cordifolia) showed significantly reduced activity (p < 0.05) Group showed the lowest GOT eflux (694.40±14.28U/L) at 72 hours. For LDH, eflux remained steady up to 48 hours (p>0.05) across all groups, followed by a sharp, significant escalation (p < 0.05) at 72 hours. Although Group 1 had the highest absolute activity at 72 hours (358.66±7.99U/L), no statistically significant variations (p > 0.05) were established among the rows at any specific time point, indicating that treatment efects on LDH kinetics were elusive.

Lipid Peroxidation (LPO) and Total Antioxidant Capacity (TAC) estimation

The biochemical markers for oxidative stress were analyzed and are given in the Tables 12.0 and 13.0 and Figures 10.0 and 11.0. Malondialdehyde generation measured via LPO, escalated over time across all groups (p < 0.05) the control group exhibited severe oxidative stress, with LPO rising to 6.52±0.03 μmol / L at 72 hours. Giloy (Tinospora cordifolia) extract administration significantly suppressed (p < 0.05) peroxide accumulation. At 72 hours, Groups 2, and efectively restricted LPO levels (µmol/L) to a range of 3.79±0.02, 3.68±0.35 and 3.58±0.45 respectively, were showing no significant diferences among themselves (p>0.05) Concurrently, Total Antioxidant Capacity (TAC) degraded progressively over time across all experimental groups (p < 0.05, Table 12). Interestingly, at all post-baseline intervals (24, 48 and 72 hours), the untreated control group (Group 1) maintained significantly higher TAC levels (p < 0.05) in the supernatant fluid compared to all the T. cordifolia-supplemented groups. By 72 hours, the control group held a TAC value of 454.33±37.04mol/L}, whereas the Giloy extract-treated groups dropped uniformly (p > 0.05) to a lower range i.e 366.67±36.64 mol/L (Group 2), 372.67±36.58 mol/L (Group 3) and 367.15±38.46 mol/L (Group 4) respectively. Lipid peroxidation's efects on the spermatozoa are numerous such as membrane damage, irreversible loss of motility, inactivation of enzymes and a high rate of leakage of intracellular sperm constituents like adenine, pyridine and enzymes, protein and DNA damage (Kadirvel et al., 2014). In the present study, levels of TAC in the preserved boar semen were 1.13±0.05 nm0l/10 8 and 1.22±0.05 nmol/10 8 spermatozoa, respectively. The TAC values obtained for preserved semen were 1.6±0.04 nmol/10 8 spermatozoa and 1.69±0.04 nmol/10 8 spermatozoa, respectively. These values did not difer significantly between groups Patel et al., 2016 (172.70±0.41).

Acknowledgement

AcknowledgmentsThe author is very much grateful to the College of Veterinary Science and A.H., Rewa, Madhya Pradesh India for providing the facilities for conducting this research.

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