Augmented oxidative stress and reduced mitochondrial function in ageing goat testis

Abstract Recently, there is a significant increase in the commercial use of goat products. Nevertheless, there are very few reports on the characterization of redox biomarkers and mitochondrial function in the goat testis. Therefore, in this study we studied the markers of oxidative stress and mitochondrial functions in the goat testis during the process of ageing. Alterations in the markers of oxidative stress/redox biomarkers (contents of reactive oxygen species, nitrite, lipid peroxide, protein carbonyl, glutathione and activities of glutathione peroxidase, monoamine oxidase) and mitochondrial function (Complex‐I and Complex‐IV activities) were elucidated during the process of ageing. Augmented oxidative stress and decreased mitochondrial function were prominent during ageing in the goat testis. Ageing can lead to induction of oxidative stress and decreased production of ATP; however, the prooxidants generated must be effectively removed from the body by the innate antioxidant defence system to minimize the damage to the host tissue. Conversely, the antioxidants cannot completely scavenge the excessive amount of reactive oxygen species produced during ageing or pathological conditions leading to significant cell death and tissue damage. Thus, the use of effective and potent antioxidants in the feed could significantly reduce oxidative stress and improve mitochondrial function, resulting in enriched goat health.

| 767 MAJRASHI et Al. 2015). Ageing also affects the reproductive system and the associated decline in reproductive capacity could result from a combination of morphological and molecular alterations in the reproductive organs (Handelsman & Staraj, 1985). Gonads, especially testis, are of particular interest in ageing research due to decline of reproductive capacity.
Furthermore, ageing of testis has direct implications for longevity, as decreased testicular function is associated with decreased longevity in several species (Partridge, Gems, & Withers, 2005). Literature on testicular function and its deficits during ageing has been, thus far, limited.
The commercial business and recreational farming associated with domestic goat (Capra aegagrus hircus, family Bovidae) is developing substantially at large numbers and the various quality goat-derived products are constantly being consumed around the world (Gall, 1996;Wilkinson & Stark, 1987). Due to the upswing in the comprehensive importance of goats, the goats must be raised healthily and have to be observed carefully to retain the well-being of an individual goat and its herd (Jaudas, Mobini, & Jaudas, 2006).
In addition to the declining reproductive function, ageing testes is associated with loss of muscle mass and concomitant increase in fat mass (Katznelson et al., 1996); decreased muscle strength (Martin, Farrar, Wagner, & Spirduso, 2000) and decreased bone mineral density (Riggs et al., 1982), all of which can adversely affect the well-being. However, the relationship between oxidative stress and testicular ageing has not been investigated. Hence, this study was designed to demonstrate the influence of ageing on the testis of kiko goats in different age groups and to assess the role of oxidative stress and mitochondrial functions in the testis during ageing.

| Chemicals and reagents
Thermo Scientific Pierce 660 nm Protein Assay reagent kit was purchased from Thermo Scientific (Pierce) for protein quantification. Griess reagent was purchased from Thermofisher Scientific.

| Goat testis tissue
Kiko goats of three different age groups were obtained from commercial vendors. The sample size considered for each group was 5 (n = 5) in number applicable to all the three different age groups.
Hence a total of 15 goats were used. A two-sided t-test was used and significance for statistical comparisons was set at p < .05. The three age groups include: Neonate (13-20 days), juvenile (108-124 days) and adult (over 6-9 months). The goats had free access to water.
Medium quality forage (>10% protein) were provided. Does were fed a pelleted supplement (16% CP, 3.04 Mcal/kg of DE, as fed) at 454 g/d from kidding to weaning. The goats were slaughtered to collect their testes. The two testes of each of the 15 goats were carefully removed, labelled for proper identification and placed in liquid nitrogen after collection until further analysis. A prober amount of the testes corpus (body) was weighed, minced separately with addition of Halt™ Protease Inhibitor Cocktail. This was followed by homogenization with a high-magnitude ultrasonic sonicator for 2 min with 2 ml PBS. The homogenate was further centrifuged at 12,000 RPM for one hour and the supernatant was collected to be used in experiments.

| Reactive oxygen species (ROS) generation
The generation of reactive oxygen species generated in the testis of neonate, juvenile and adult groups was estimated via spectrofluorometry by measuring the conversion of non-fluorescent chloromethyl-DCF-DA (2′, 7-dichlorofluorescindiacetate, DCF-DA) to fluorescent DCF using an excitation wavelength of 492 nm and an emission wavelength of 527 nm. Results were expressed as percentage change from the control (Dhanasekaran, Tharakan, & Manyam, 2008;Katz et al., 2017;Zheng et al., 2014).

| Nitrite content
Nitrite content in the testis of neonate, juvenile and adult groups was measured using Griess reagent. An azo product formed was measured spectrophotometrically at 545 nm (Giustarini, Dalle-Donne, Colombo, Milzani, & Rossi, 2008).

| Mitochondrial Complex-I activity
NADH oxidation to NAD⁺ is catalysed by mitochondrial Complex-I (NADH dehydrogenase). Tissue homogenate obtained from the testis of neonate, juvenile and adult groups was added to PBS and conversion of NADH to NAD⁺ was measured spectrophotometrically at 340 nm (Bhattacharya et al., 2018;Ramesh et al., 2018;Ramsay, Dadgar, Trevor, & Singer, 1986).

| Glutathione peroxidase activity
Spectrophotometric method was used to measure glutathione peroxidase activity in the testis of neonate, juvenile and adult groups using NADPH as a substrate (Ahuja et al., 2017;Majrashi et al., 2018).

| Protein quantification
Protein was quantified using Thermo Scientific Pierce 660 nm Protein Assay reagent kit (Pierce, Rockford, IL). Bovine serum albumin (BSA) was used as a standard for protein measurement.

| Statistical analysis
Data were reported as mean ± SEM. Statistical analyses were accomplished using one-way analysis of variance (ANOVA) followed by Dunnet's multiple comparisons test (p < .05) and was determined to be statistically significant. The statistical analyses were performed using Prism-V software (La Jolla, CA, USA). All the determinants were made in triplicates until otherwise mentioned.

| RE SULTS
General data: The average body weight of neonates was 3.06 ± 0.43 kg, juvenile was 19.5 ± 3.12 kg and adult age group was 43 ± 5.78 kg. No change in food consumption habits or any abnormal behaviour was noted. No statistically significant weight loss or any serious illness was noted in all the goats from different age groups.

| Ageing increases reactive oxygen species and nitrite in goat testis
The generation of reactive oxygen species (ROS) triggers oxidative stress and induces irreversible oxidation of lipids and proteins, which has lethal effects on cells leading to cell death. Statistically significant increase in ROS generation was noted in both juvenile (p = .003) and adult (p = .02) goat testis when compared with neonate group

| Ageing inhibits mitochondrial function in goat testis
To explore the effects of ageing on mitochondrial function and to understand the molecular processes involved, we evaluated Complex-I and Complex-IV activity in three different groups. Ageing notably decreased mitochondrial function, as demonstrated by statistically significant decrease in Complex-I activity in the juvenile (p = .035) and adults (p = .01) when compared with the neonate group (n = 5, p < .05; Figure 2a). However, our results showed that ageing did not affect Complex-IV activity (n = 5, Figure 2b).

| Ageing alters the activities of antioxidant enzymes in goat testis
A defence mechanism of the cell is to promote antioxidant expression and activity, which protects against highly reactive oxy or nitro radicals and their harmful toxic effects. We therefore

| Ageing increases lipid peroxidation and protein carbonyl in goat testis
The generation of free radicals triggers oxidative stress and induces irreversible oxidation of lipids and proteins. Therefore, lipid peroxidation and protein carbonyl content were investigated in this study. Ageing significantly induced lipid peroxidation in juvenile

| Ageing alters monoamine oxidase activity in goat testis
The mitochondrial location of MAO has also been reported (Lehninger, 1975), and MAO is used as a marker enzyme to indicate the presence of the outer membrane in the mitochondria. Ageing significantly decreased monoamine oxidase activity in the juvenile F I G U R E 1 Statistical comparisons were made using one-way ANOVA/Dunnett's multiple comparison test. Note (*) indicates a statistically significant difference when compared with controls. (a) Effect of ageing on ROS generation: ROS was measured spectrofluorimetrically. Ageing showed a significant increase in ROS generation (*p < .05, n = 5). ROS was measured as relative fluorescence units (492/527 nm)/mg. Results are expressed as (%) change as compared to the control ± SEM. (b) Effect ageing on Nitrite content: Nitrite content was measured spectrophotometrically. Ageing showed a significant increase in Nitrite content (*p < .05, n = 5). Nitrite content was measured as the absorbance of chromophoric azo product (545 nm) resulting from the reaction of NO 2 with sulfanilamide under acidic condition. Results are expressed as (%) change as compared to the control ± SEM

| D ISCUSS I ON
Age-dependent elevation in mitochondrial oxidative stress can contribute to decrease in testicular function. In this study, we report that ageing increased markers of oxidative stress/redox biomarkers such as ROS and nitrite, decreased mitochondrial complex activity, complexes increase mitochondrial ROS production (Murphy, 2009).
In our study, we found a statistically significant reduction in Complex I activity in juvenile and adult goat testis with no significant changes in Complex IV activity. Mitochondrial ROS formation is inversely related to Complex I activity which might indicate that the mitochondrial ROS generation is primarily due to compromised Complex I, and to a lesser extent in Complex IV (Zorov, Juhaszova, & Sollott, 2014).
Mitochondrial electron transport is an important subcellular source of ROS. Complex I, also, is an integral membrane complex of the  (Hansford, Hogue, & Mildaziene, 1997).
The produced superoxide is then scavenged by the mitochondrial enzyme superoxide dismutase to produce H2O2. Therefore, a deficient complex I activity can be considered a potential source of ROS in ageing tissues (Zalewska, Ziembicka, Żendzian-Piotrowska, & Maciejczyk, 2019). Although ROS production may not be critical factor for ageing (López-Otín et al., 2013), they are more likely to exacerbate age-related diseases progression via oxidative damage and interaction with mitochondria (Dias, Junn, & Mouradian, 2013 (Zuo, Hemmelgarn, Chuang, & Best, 2015). We found a statistically significant decline in antioxidant (glutathione) levels. A decline in antioxidant system leads to increased susceptibility to oxidative stress especially in elderly as there is a decline in the efficiency of the endogenous antioxidant systems. Hence, organs with high rates of oxygen consumption and limited respiration levels such as brain and heart, are highly susceptible to this phenomenon.
This partially explains the high prevalence of cardiovascular diseases and neurological disorders in the elderly (Corbi et al., 2008). Similarly, the decline in antioxidant system in the testis could account for the decline in sexual and reproductive capacities. Interestingly we found an increase in the glutathione peroxidase activity in juvenile and adult goat testis which we believe could be attributed to the compensatory response.
The altered prooxidant-antioxidant redox status is more likely triggered by net effect of low antioxidants and increased reactive oxygen species (Chung et al., 2009;Lennicke, Rahn, Lichtenfels, Wessjohann, & Seliger, 2015). An imbalance between prooxidant-antioxidant redox status and the dysregulation of the immune system as seen in ageing may lead to the exaggerated systemic inflammatory response with activation of inflammatory mediators. Hence, chronic inflammation as seen in ageing may serve as a pathophysiologic association which converts normal functional changes to the age-related degenerative diseases (Viola & Soehnlein, 2015).
This increase in the reactive oxygen species in the juvenile and adult goats may be associated with the innate (non-specific) immune system. The respiratory burst associated with the innate immunity is a process that involves enzymes and produces several types of reactive oxygen species. The reactive oxygen species essentially impact cellular processes by affecting the cell signalling, transcription factor and translation regulator under the controlled physiological conditions which in turn affects the cell growth. However, F I G U R E 3 Statistical comparisons were made using one-way ANOVA/ Dunnett's multiple comparison test. Note (*) indicates a statistically significant difference when compared with controls. (a) Effect of ageing on glutathione content: Glutathione content was measured spectrophotometrically.
Ageing showed a significant depletion in GSH content (*p < .05, n = 5). Results are expressed as (%) change as compared to the control ± SEM. (b) Effect of ageing on glutathione peroxidase activity: Glutathione peroxidase activity was measured spectrophotometrically using NADPH as substrate. Ageing showed a significant increase in glutathione peroxidase activity (*p < .05, n = 5).
Results are expressed as (%) change as compared to the control ± SEM  (Hauck & Bernlohr, 2016). In our study, we found a statistically significant increase in lipid peroxidation and protein carbonyl content in juvenile and adult goat testis. Lipid peroxidation has shown to affect the sperm survival and the sperm fertility (Guthrie & Welch, 2012).
Lipid peroxidation-induced decreased sperm motility is because of the reactive oxygen species-induced lesion in ATP utilization or in the contractile apparatus of the flagellum and can result in testicular hypoplasia and infertility.
The mitochondrial theory of ageing is still deliberated as an extension of the free radical hypothesis. Given the close relationship between oxidative stress, inflammation and ageing, the oxidation-inflammatory theory of ageing has been proposed: ageing is a loss of homeostasis due to a chronic oxidative stress. The consequent activation of the immune system induces an inflammatory state that creates a vicious circle in which chronic oxidative stress and inflammation feed each other, and consequently, increases the age-related morbidity and mortality (Fuente & Miquel, 2009

| CON CLUS ION
Health and lifespan of goats has been related to oxidative stress and mitochondrial functions. There is a constant increase in the prooxidants and decreased antioxidants & ATP contents during ageing in goat testis. The results from this study clearly established the altered redox status and mitochondrial dysfunction during ageing in goat testis. Herein, we provide a strong basis for future mechanistic studies and therapeutic strategies to improve the well-being of goats.

ACK N OWLED G EM ENTS
We sincerely thank the Department of Drug Discovery and