Curcuma xanthorrhiza Diluent’s Effect on the Freezing and Thawing of Thin-Tailed Ram Sperm

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INTRODUCTION
I n recent years, artificial insemination (AI) technology has often been used in Indonesia. Artificial insemination limits the spread of infectious illnesses when organisms are near or share the same environment. Sperm Cryopreservation one of AI procedures, enables the broad transmission of significant genetic material, which also enhances the spring number in livestock for a short time. However, the application of frozen sperm for artificial insemination is hampered by poor fertility rates in sheep after the treatment or protocol. Freezing and thawing of spermatozoa is followed with diminished cell motility, viability, and fertility (Atessahin et al., 2008;Al-Mutary, 2021). Biochemical and physical alterations in spermatozoa membrane caused by oxidative damage during the freezing process can also interfere with spermatozoa function and even cause cell death. Furthermore, it is known that during the freezing process, it has the capacity to create lipid peroxide and reactive oxygen species (ROS). Which cause a deterioration in the quality of spermatozoa via a redox mechanism (Peris-Frau et al., 2020). ROS damage spermatozoa DNA by exposing sperm cells at high oxygen pressure and lowering sperm quality such as motility and viability (Jumintono et al., 2021). ROS-induced oxidative stress in oxygenated sperm caused by oxygen-containing ROS can damage cells and tissues. Spermatozoa are highly sensitive to oxidative stress because of their high fatty acid content, which is not balanced by adequate antioxidant levels. Antioxidants neutralize ROS and protect cells from O n l i n e F i r s t A r t i c l e oxidative damage; hence, sperm with high ROS levels have increased motility, concentration, and morphology, and reduced DNA damage (Jumintono et al., 2021). A fast reduction in temperature and pressure during the freezing process also causes physical and chemical damage to the spermatozoa membrane (Aitken and Drevet, 2020;Jumintono et al., 2021). Overall, the whole process that occurs during the preparation of frozen semen results in damage to sperm cells both due to physical factors (the freezing process that occurs quickly) and chemically (the formation of ROS during the process) which directly reduces the quality of frozen semen produced. Therefore, the addition of certain antioxidant substrates is needed to inhibit or reduce the negative impact of the sperm freezing process (Priyanto et al., 2023). Curcuma xanthorrhiza rhizome, often known as Javanese turmeric or temulawak (Family: Zingiberaceae), is a commonly used as herbal cure and potentially have antioxidant function (Pujimulyani et al., 2018). The primary constituents of C. xanthorrhiza include starch (50-60%) and volatile oils (3-12%), including phelandren, camphor, tumerol, sineol, borneol, xanthorrhizol (1.48-1.63 %), sesquiterpenes (-curcumene, ar-curcumene, bisabolane, lactone germacone), and flavonoids (Rohman et al., 2020). C. xanthorrhiza rhizomes contain a plethora of the active compounds terpenoids and curcuminoids (Zhang et al., 2014;Rahmat et al., 2021) which has antibacterial and antioxidan effects (Rosidi et al., 2016;Widyastuti et al., 2021). DPPH (1,1-diphenyl-2-picrihydazine), superoxide anion, iron-reducing antioxidant power (FRAP), and metal bonding activity were used in previous investigations to determine C. xanthorrhiza's antioxidant capacity (Devaraj et al., 2014). According to the experiment's findings, curcumin, which contains phenolic and methoxy groups on its phenyl rings and diketone moiety, is an active component of C. xanthorrhiza's antioxidant function (Masuda et al., 2001;Priyadarsini, 2014). As is well known, curcumin blocks free radicals by neutralizing lipid peroxyl beforehand, which threatens membrane PUFA (Rahmat et al., 2021). These natural chemicals or compounds may be termed antioxidants because of their activities as (i) scavengers of reactive species, (ii) chelators of heavy metal ions such that the metals are unable to form reactive species, (iii) quenchers of singlet oxygen, (iv) breakers of free radical chain reactions, and (v) reducing agents (Rohman et al., 2020). The search for natural compounds with significant antioxidant activity seems to be a helpful preventative approach for phenotypes associated with free radicals (Jakubczyk et al., 2020), therefore there are emerging increasingly restrictive rules on the use of chemical antioxidants that have the potency to damaging the environment in the manufacturing process.
A substance of C. xanthorrhiza was not previously employed in ram sperm diluent cryopreservation. The present research was conducted using thin-tailed ram sperm to evaluate the cryoprotective advantages of C. xanthorrhiza as a semen diluent due to its widespread availability in tropical environments and its potential antioxidant properties. The main objectives of this study were to standardize the optimal dose of C. xanthorrhiza to be added to the semen extender and to evaluate the effects of adding C. xanthorrhiza as a semen diluent on cryocapacitation associated and apoptotic-like alterations in spermatozoa after equilibration and freezing-thawing.

Animal experimental design and semen collection
Four mature and heathy thin-tailed rams (35-40 kg body weight; 2-5 y.o) were employ in this research. Rams have been kept at the Physiology and Reproduction Laboratory, Faculty of Animal Science, Gadjah Mada University, which is then inspected for livestock health and found to be clinically free of infectious diseases and external or internal parasites. The ejaculates were collected at 7-8 am twice weekly, and rams were regularly used for semen collection before (Gupta et al., 2022). All rams were individually fed the same concentrate mixture (CP 16%; 2.8%/BW), 10%/BW forage, and kept in individual pens. Since there were no invasive assessments of the animals, ethics committee approval was not necessary for this research.

Semen processing and evaluation
The quality and profile of the sperm was analyzed shortly after collection such as motility, viability, pH, sperm concentration, odor and semen colour to determine whether this fresh semen was appropriate to be used as a freezeable semen raw material. A Neubauer hemocytometer was used to examine the quality of fresh semen. Fresh sperm (100 μL) was positioned on a glass slide, examined under the microscope, and graded on a rating system ranging from 0 (no motility) to 100 (excellent motility), the assessment process was conducted by three experts who were experienced in assessing sperm quality to ensure the objectivity of the assessment. Spermatozoa viability (%) and abnormalities (%) were defined using a fixed smear stained using the eosin-nigrosin method. Nigrosin enhances the differentiation between the surrounding area and the sperm's head, rendering it easier to make observations. Eosin dyes just deceased sperm, rendering them a dark pink color, while the living ones appeared in white colours or do not absorb eosin. The proportions of both alive and death sperm were determined, and all O n l i n e

F i r s t A r t i c l e
primer or secondary sperm abnormality could also be determined (El-Zeftawy et al., 2020). This study analyzed semen with > 80% progressive motile spermatozoa and > 90% viability for subsequent examination. Yolk citrate (2.9% (v/v) sodium citrate dihydrate, 100 ml aquadest mixed with egg yolk 20%, 8% (v/v) glycerol (Merck, Germany), 1.000 IU/ml Penicillin and streoptomycin as antibiotics was used as the base semen diluent (freezing extender). The semen was dissolved with diluent until it reaches 50 mg/mL. Five equal aliquots of pooled ejaculate were divided and diluted (37°C) with diluent containing C. xanthorrhiza (1%, 2%, 3%, 4% called P1, P2, P3, and P4) and a base diluent as a control (P0). Then the fresh diluent semen was evaluated as the before frozen quality. The straws were afterwards adjusted at 5°C for 4 h. The equilibrated semen was collected and encapsulated to 0.25 mL straws. Straw had been placed in nitrogen vapor 5 cm over liquid nitrogen for ten minutes, and sperm was stored in liquid nitrogen. Following being stored for 24 h, each of the straws were independently thawed for semen assessment. subsequently freezing, each sample of sperm was promptly evaluated for its sperm quality. This study compared the efficacy of sperm before and after freezing in order to evaluate the effects of C. xanthorrhiza extract supplementation thoroughly.

Semen evaluation
The data observed were the concentration, motility, viability abnormalities, and plasma membrane integrity/ HOST of the spermatozoa before and after freezing. Thawing procedure was conducted by applying warm water to the frozen semen straw for 10 min until the semen is completely thawed. The evaluation of thawed sperm was conducted in the same manner as the evaluation of raw sperm described above. The hypoosmotic swelling test was conducted through exposing 0.1 ml of semen with 1 ml of 150 M hypoosmotic solvent for 30 min at 37°C. Following incubation, 0.2 ml of the solution was disseminated employing a cover sheet on slide glass. A fluorescent field microscope was utilized to investigate 200 spermatozoa at a magnification of 1000x. It has been observed that spermatozoa have a protruding or curved tail. This test measures the functional integrity of the sperm membrane. (Bucak et al., 2012).

Statistical analysis
The outcome variables are represented as the mean standard error of the mean (Mean±SEM). The influence of C. xanthorrhiza extract administration in each circumstance before and after freezing procedure was determined through a multifactorial analysis of all data. In addition, the results for each condition were analyzed using one-way analysis of variance followed by Tukey's post hoc test to determine whether or not there were statistically significant differences in all parameters among the groups. In a regression evaluation, a predictive equation was established with the dependent variable adjusted supplementation and the independent variable sperm quality. For all statistical analyses, SPSS statistics software (version 26.0; IBM Corp., Chicago, IL, USA) was utilized (Kumaresan et al., 2020).

RESULTS
In five distinct experiments, the implications of addition C. xanthorrhiza on sperm qualities prior to and following freezing and thawing were determined. All qualities parameters such as motility, viability, abnormalities, and plasma membrane integrity/HOST (%) of sperm from thin-tailed rams supplemented with different dosages of C. xanthorrhiza (Temulawak) were better than the control P0 (Table I). The addition of 3% C. xanthorrhiza extract resulted in the most outstanding motility both in freezing treatment (76.14±1.35% total means with different superscripts within a row differs significantly (p<0.05). *total means with different superscripts within a row differs significantly (p<0.05). P 0 , without C. xanthorrize; P 1 , 1% of C. xanthorrize; P 2 , 2% of C. xanthorrize; P 3 , 3% of C. xanthorrize; P 4 , 4% of C. xanthorrize.

O n l i n e F i r s t A r t i c l e
pre-freezing; 60.00±1.63% after thawing) and viability percentage (85.00±2.00% pre-freezing and 70.14±1.57% after thawing) p<0.05). The data showed significant differences in pre-freezing and post-thawing in ram semen diluted with C. xanthorrhiza at different concentrations, as shown in Table I.

Effect of C. xanthorrhiza on semen quality
The data showed that addition of Curcuma xanthorrhiza 3% resulted in the highest motility in both freezing treatments, which is supported by earlier studies showing that curcumin (the antioxidant active ingredient of C. xanthorrhiza) has similar effects, which increased semen quality after the addition of curcumin in cattle (Gupta et al., 2022), bull (Tvrda et al., 2012(Tvrda et al., , 2016, buffalo (Herbowo et al., 2019), rams and rats (Soleimanzadeh and Saberivand, 2013;Omur and Coyan, 2016), goat (Bucak et al., 2010), and boars (Chanapiwat and Kaeoket, 2015). However, administration of C. xanthorrhiza to any species, notably goats, has never been conducted in an earlier study.
Sperm motility is crucial for the process of transporting spermatozoa to the ovum and is a significant factor in spermatozoa penetrating the cumulus cells and zona pellucida of the ovum for fertilization (Bucak et al., 2012). In this study, C. xanthorrhiza addition significantly enhanced the motility and viability spermatozoa compared to the control (P<0.05) in both frozen and thawed semen, which is consistent with other studies in cattle and goats using diluent containing curcumin (Bucak et al., 2012;Kumar et al., 2014). Curcumin addition in pre-freezing and post-thawed ram spermatozoa markedly improved CASA motility and viability. The present investigation determined which the addition of C. xanthorrhiza to sperm diluent effectively maintains more than 40% of the sperm's post-thawing motility (PTM), which is suitable for insemination (Table I). This was due to curcumin improved capacitation and the acrosome response, both of which enhance sperm motility in human sperms. This was accomplished by increasing the amount of the transcriptional nuclear component Nrf2 (Nuclear factor E2-related factor 2), which is an inducible transcription factor essential for maintaining redox signal transmission against oxidative stress in spermatogonia and sperm cytoplasm in mammals (He et al., 2022). The bZIP protein NRF2 controls the regulation of promoters expressing compounds involved in detoxifying ROS to shield cells from oxidative damage, Nrf2 induces the transcription of a series of antioxidant enzymes directly dependent on the antioxidant response element (ARE) promoter (Aydos et al., 2021). However, the effects on small ruminants have not been clarified yet. The only conclusion that can be drawn from this study is that the antioxidant activity of curcumin present in C. xanthorrhiza actively minimizes the detrimental effects of ROS, which means that it will generally maintain sperm quality, especially after thawing (Santonasato et al., 2021). Curcumin and xanthorhizol (XNT) in C. xanthorrhiza acted as antioxidant agents to suppress the growth of ROS (Carapina da Silva et al., 2019), which could maintain sperm quality in this study. This preservation due to XNT decreased the oxidative damage caused by free radicals (Lim et al., 2005). XNT antioxidant characteristics demonstrate powerful neuroprotective benefits by inhibiting hydrogen peroxide (H 2 O 2 )-induced lipid peroxidation, glutamate-induced neurotoxicity, and ROS production in cells. Additionally, the presence of a phenolic hydroxyl group (sesquiterpene phenol) on the bisabolene skeleton of XNT likely contributed to its powerful antioxidant capabilities by chelating Cu 2+ . This may inhibit the onset of LDL oxidation and the production of free radicals inside the lipoprotein (Jantan et al., 2012;Oon et al., 2015), and depress cell death in this study. According to this study, curcumin acts as a potent antioxidant that reduces the consequences of oxidative stress. This is supported by a previous study using the DPPH method (Rosidi et al., 2016), which evaluated the antioxidant activity of the ethanol extract of C. xanthorrhiza using the liquid-liquid extraction technique in hexane. With an IC 50 value of 86 ppm, the C. xanthorrhiza extract was shown to have a reasonably potent antioxidant effect. It reduces oxidative damage through engaging with various biochemical processes, such as its capacity to neutralize metals that are toxic and modulate the functions of a large number of enzymes (Jakubczyk et al., 2020). Moreover, curcumin which also cointain in C. xanthorrhiza has a higher antioxidant capacity than vitamins E and C, and beta-carotene (Rosidi et al., 2016;Septiana et al., 2020). Curcumin is expected to be used as a semen supplement because of its capacity for antioxidants, as indicated by earlier research. Curcumin ingredients in C. xanthorrhiza diluent semen scavenge ROS and suppress LPO, enhancing semen quality through reducing the oxidative damage in sperm cells (Bucak et al., 2012;Tvrda et al., 2012).
The lack of antioxidants in order to decrease the amount of the generation of ROS resulted in decreased motility and viability in the control group. This results in the acrosome response affecting the sperm's lifespan, sperm create and export ROS to the extracellular environment, most of which are produced by mitochondria in response to flagellar activity and leads early sperm maturation and derivate the fertilization sperm ability or accelerate sperm death (Riesco et al., 2021). Under normal circumstances,

O n l i n e F i r s t A r t i c l e
Curcuma xanthorrhiza Diluent's Effect on Ram Sperm 5 naturally produced antioxidants or antioxidant enzymes protect sperm in a decreasing microenvironment. Elevated oxidative stress causes modifications in lipids, proteins, and nucleic acids, which ultimately contribute to a decrease in mitochondrial function and cell mortality (Tan et al., 2018;Jakubczyk et al., 2020). C. xanthorrhiza suplementation had a better protective impact on functional membrane integrity than the control both in pre-freezing and after thaw semen, although not significantly different (P>0.05). Additionally, the data showed that the addition of C. xanthorrhiza led to a higher HOST score, although it was not significantly different (p>0.05) from that of the control. These characteristics are linked to the control of proinflammatory cytokines, nitric oxide synthase (iNOS) enzymes, cyclooxygenase-2 (COX-2), lipoxygenase, xanthine oxidase, and reduction of malondialdehyde (MDA) (Alizadeh and Kheirouri, 2019;MS et al., 2020).
The addition of 3% C. xanthorrhiza (P3) resulted in a slight preservation of the percentage of membrane integrity, motility, and viability both in pre-and post-thawing semen in all groups. This demonstrated that the addition of up to 3% C. xanthorrhiza extract substantially preserved the characteristics of thin-tailed ram spermatozoa prior to and after thawing. In the presence of curcumin, cysteine, or hyaluronan in the cryogenic state, there was no reduction in MDA levels in ram and goat spermatozoa (Bucak and Tekin, 2007;Bucak et al., 2008Bucak et al., , 2010. This study found an interesting data that supplementation with 4% C. xanthorrhiza (P4) resulted in reduced sperm quality. An intriguing finding in this study was that sperm quality metrics decreased after freezingthawing with the addition of 4 % C. xanthorrhiza, which is in line with a previous study showing a detrimental impact of curcumin at high concentrations on these parameters (Gupta et al., 2022). After freezing and thawing, excessive curcumin in C. xanthorrhiza contain in semen may promote sperm intracellular acidification and hyperpolarization of the membrane, which may explain why sperm activity is reduced (Naz, 2014). The potential of acidification within cells and sperm metabolism damage cannot be ruled out. C. xanthorrhiza and their curcumin amounts and reactions may be influenced by organisms, the sort of semen enhancer used, the method of processing of the semen, and freezing techniques (Tvrdá et al., 2016). However, curcumin has both negative and positive effects on sperm function, according to the amount present in the sperm extender (Naz, 2011). Therefore, it is essential to note the formulation with curcumin added to the diluent. Regression test using cubic curve showed the R 2 values on motilities and viabilities had higher than 0.85 and could be used as a predictor on the higher addition of C.

xanthorrhiza.
Effect of pre-freezing and after thawing on semen diluted with Curcuma xanthorriza Cryopreservation or freezing of sperm is one of the most crucial techniques for advancing bioprocesses for assisted reproduction (Yánez-Ortiz et al., 2021). This study found that all parameters after thawing showed reduced sperm quality due to the cryopreservation mechanism (Table I). Cryopreservation-induced oxidative stress caused by free radicals is the primary cause of apoptosis in sperm cells (Gupta et al., 2004(Gupta et al., , 2022. Previous investigations have shown that spermatozoa undergo apoptotic-like changes when frozen and thawed (Martin et al., 2004), including decreased mitochondrial transmembrane potential, chromatin crosslinking, and DNA fragmentation, all of which lead to decreased viability and motility. Pre-post thaw, there is a nearly two-fold increase in apoptosis-like spermatozoa, which lowers the sperm quality (Shah and Andrabi, 2021). The freeze-thaw process targets sperm DNA and the protamine process. A previous study found that freezing-thawing of goat spermatozoa increased DNA fragmentation. DNA fragmentation was evident in chromatin dispersion (the halo surrounding the nucleus) and loss of protamine, which we observed in the aberrant sperm cell population in this investigation (deprotamination). DNA fragmentation in sperm cells is associated with increased deprotamination, which increases the risk of infertility (Kritaniya et al., 2020). C. xanthorrhiza addition of up to 3 % in this study was shown to protect sperm quality throughout the freezing-thawing process, and this study's findings support other studies (Tvrdá et al., 2016). Curcumin was previously added to the semen extender in bull spermatozoa, and similar effects on the mitochondrial transmembrane potential were observed (Tvrda et al., 2012). The antioxidant curcumin protects sperm mitochondria from oxidative stress, resulting in sperm mitochondrial function and protection (Omur and Coyan, 2016). At higher doses, C. xanthorrhiza did not preserve sperm DNA, protamine, or membrane phosphatidylserine, which may have been related to curcumin toxicity at higher concentrations (> 3 %). This finding is similar to that of another study that used curcumin as semen diluent (Kumaresan et al., 2020). This is further supported by the graph below, which shows a regression trend for motility (R 2 = 0.95; P = 0.055) and viability (R 2 = 0.88; P = 0.097) after thawing ( Fig. 1; Table  I). Consequently, discovering and utilizing the optimal concentrations of C. xanthorrhiza in the extender at 3 % might help protect ram sperm from harm throughout the cryopreservation storage procedure.

O n l i n e F i r s t A r t i c l e
S. Bintara et al.
In conclusion, the supplementation of 3 % C. xanthorrhiza to the semen diluent significantly enhanced pre-post thaw semen quality in thin-tailed ram semen by reducing free radical-mediated oxidative stress. The overall improvement in the feezability of ram sperm was attributable to a reduction in cryocapacitation-associated alterations, apoptotic-like changes, and protein carbonyls following freezing-thawing. C. xanthorrhiza demonstrated dual effects, improving sperm quality at lower doses (up to 3 %) and degrading spermatozoa at higher doses (≥ 4 %); consequently, the amount of C. xanthorrhiza must be considered prior to its use as an additive.

IRB approval
Not applicable.

Ethics statements
There were no invasive assessments of animals, and ethics committee approval was not necessary for this study.

Statement of conflict of interest
The authors have declared no conflict of interest.