Goat sperm are susceptible to freeze–thawing [
17] due to the extreme temperatures and osmolarities change [
18]. Cryoprotectants are needed by spermatozoa to survive during the freeze–thawing process. Ideally, cryoprotectants have a low molecular weight to penetrate cell membranes. Glycerol is an intracellular cryoprotectant, thereby preventing the ice crystals formation, which could lead to avoiding the rupture of the membrane [
19].
4.1. GTE Dose
Without GTE addition, the post-thawed semen quality of the T0 group had the lowest sperm viability, motility, and IPM than the other groups. As mentioned earlier, the goat sperm is sensitive to oxidative stress. The cryopreservation process of semen leads to cold shock of sperm, which changes the ratio of polyunsaturated fatty acids (PUFA) and lowers the cholesterol content, causing instability of the sperm membrane. Plasma membrane integrity is essential for protecting the organelles of sperm and molecular transportation; thereby, it is crucial for sperm viability and sperm motility [
20]. Sperms had antioxidant enzymes, such as glutathione peroxidase (GPX), catalase (CAT), and superoxide dismutase (SOD). However, the small volume of the sperm cytoplasm makes the transfer of these enzymes to the other part of the sperm challenging [
21]. The concentration of antioxidant enzymes also decreased with the extender dilution. The unresolved oxidative stress causes a decrease in sperm motility and sperm viability [
22]. Without GTE’s addition, the T0 group had the highest MDA level and DNA fragmentation of the groups. The high ROS generated freeze–thawing causes the peroxidation of double bonds of docosahexaenoic acid in PUFAs and produced MDA, which is a toxic lipid aldehyde species [
23]. The high ROS may also lead to DNA fragmentation mediated by lipid peroxidation, modification of 8-hydroxy-guanine or 8-hydroxy-20-deoxyguanosine on mitochondrial and nuclear DNA base, destabilizing the DNA structure [
24]. Sperm DNA is bound to protamine in a compact state, which helps protect the DNA against these destructive processes. Protamine forms disulfide bonds, making the sperm’s nucleus resistant to physical and chemical influences [
25]. Protamine can condense and compact DNA to protect sperm from free radicals [
26]. However, the interchain disulfide bridge opening on protamines during freeze–thawing may damage sperm DNA [
27].
Post-thawed fertile sperm should have an intact plasma membrane to ensure the sperm viability, motility, and intactness of its DNA [
28]. Treatment on semen of many species revealed varies results. Study on bull sperm indicated that the using of single-layer centrifugation selected spermatozoa resulted in less DNA damage [
29]. Meanwhile, the addition of prostatic fluid to canine spermatozoa resulted in a higher proportion of spermatozoa with DNA damage after freezing–thawing [
30]. Antioxidant administration can significantly decrease oxidative DNA damage [
31]. GTE reserves the plasma membrane from lipid phosphorylation [
32], including the acrosomal cap membrane necessary for the acrosomal reaction. During the freezing process, acrosome ruin and the partial displacement of the outer acrosomal membrane with the decimation of acrosomal enzyme content commonly occur due to ice crystal formation. This change causes swelling and rupture of the sub-acrosomal region. Changes in osmotic pressure will damage the lipid membrane structure, causing stress changes in proteins channel and ion leakage of the plasma membrane. This resulted in the cytoplasmic organs’ morphological changes and opened DNA to ROS attack [
33]. The T1 group (with the addition of 0.05 mg GTE/100 mL extender) was of higher post-thawed semen quality than those of the CG. Green tea polyphenols may improve semen quality by reducing ROS production [
34], lipid peroxidation, protein carbonylation, and DNA damage, thereby improving semen quality [
35]. However, the number of these antioxidants is insufficient to offset the overproduction of ROS during freeze–thawing. The post-thawed semen quality of T1 was lower than that of T2.
The T2 group (added 0.1 mg GTE/100 mL extender) showed the highest post-thaw semen quality with the highest sperm viability, sperm motility, IPM, and lowest MDA levels and DNA fragmentation. This result is consistent with this finding that GTE’s addiction to an extender maintained the motility, viability, IPM, and DNA integrity of boar semen [
36] and Simmental bull sperm [
15]. On the same breed of goat (Kacang buck), the post-thawed semen quality of the T2 group was better than our ongoing study using AndroMed
® (minitube), namely 61.67 ± 0.29 vs. 46.19 ± 0.04% for sperm viability, 60.50 ± 0.26 vs. 40.83 ± 0.04% for sperm motility, and 58.58 ± 0.57 vs. 27.40 ± 0.04% for IPM, respectively. The EGCG, a bioactive substance of GTE, enhances post-thaw microscopic parameters and the fertility potential of buffalo spermatozoa [
8]. The advantage effects of EGCG are due to the polyphenolic groups as antioxidant and chelating agents, whereas flavonoids act as catalysts on the membrane function [
32]. Furthermore, the EGCG decreases oxidative stress parameters in spermatozoa [
8]. Green tea polyphenols act on the adenosine monophosphate-activated protein kinase (AMPK), cyclic adenosine monophosphate (cAMP), calcium ions [
34], ferrous iron, ferric iron, and low-density lipoprotein signaling pathways [
35].
The T3 group (addition of 0.15 mg GTE/100 mL extender) showed a lower post-thawed semen quality than T2. Physiologically, sperm require low quantities of ROS for the healthy functioning of sperm [
37]. The changes in ROS or antioxidant levels cause a redox imbalance. Excessive ROS or excessive antioxidants can disrupt the balance state [
38]. High doses of GTE (T3 group) cause low levels of ROS and are inadequate for normal sperm function. Higher exposure to antioxidants leads to an antioxidant paradox that significantly reduces male fertility [
31].
4.2. The Equilibration Period
The equilibration period is the duration of sperm contact with a cryoprotectant before freezing [
39]. The equilibration stage affects the sperm’s ability to adapt to the extender environment to maintain homeostasis, osmotic tolerance, and cryo tolerance and prevent physical damage, osmotic stress, and cold stress during freezing [
40]. Pre-freezing equilibration helps the sperm reach an osmotic equilibrium following the addition of the cryoprotectant. As mentioned earlier, the equilibration period of Kacang buck semen has not been established yet. Compared to other livestock species, the standard equilibration period for bull sperm before cryopreservation is 3–4 h to maintain sperm membrane integrity and motility [
41]. In Jamunapari buck semen, a two or three-hour equilibration period resulted in the best post-thawed sperm [
42]. Ahmad et al. reported that Beetal buck sperm survival is higher when equilibrated for 2–8 h [
43].
The quality of post-thawed Kacang buck semen equilibrated for one hour was better than two hours for T1, indicating that extending the pre-freezing equilibration period did not improve the quality of post-thawed semen. However, this is consistent with another report that the prolonged pre-freezing equilibrated for 24 h of ram semen was not improved in its quality [
44], and semen quality was improved neither in vitro nor in vivo [
45]. Extending the equilibration period precisely reduces the quality of Kacang buck semen after thawing. Concerning antioxidants, when equilibrated for two hours, the sperm was excessively exposed to GTE’s antioxidants for more than the one-hour equilibration period, which caused a balance shift to reductive stress. Oxidative stress and reductive stress also harm sperm fertility [
46]. Physiologically, sperm require low levels of ROS for normal functions such as sperm maturation, hyperactivation, acrosome reaction, and sperm–oocyte fusion [
37]. ROS play an essential role in sperm fertility acquisition and tyrosine phosphorylation, sterol oxidation, and cholesterol outflow during the fertilization process [
23]. Reductive stress is the powerful facet of redox imbalance that can harm sperm function. Decreased oxidative phosphorylation complex protein expression associated with mitochondrial dysfunction causes reductive stress [
35].
A dose of 0.1 mg of GTE per 100 mL of skim milk–egg yolk (SM-EY) extender had the best effect on the quality parameters of Kacang buck semen. Changes in sperm viability, sperm motility, and IPM tendencies were reduced from pre-freezing to post-thaw and from 1 to 2 h equilibration. Meanwhile, the MDA concentrations and DNA fragmentation trends increased (
Table 2). When equilibrated for one hour, the sperm motility of T2 was more than 40% (44.00% ± 0.19%), and its DNA fragmentation did not exceed 7% (6.43% ± 0.13%) (
Table 2). This result was lower than that of Simmental bull sperm diluted in a 0.1 mg GTE per 100 mL SM–EY extender, which obtained 69.17% ± 1.4% motility and 3% ± 0.5% DNA fragmentation [
15]. This difference indicated that Kacang buck sperm were more susceptible to oxidative stress than Simmental bull sperm. This result was also lower than the best quality of post-thawed Kacang buck semen diluted in an egg yolk–citrate extender supplemented with 2.5 mg of bull seminal plasma protein per milliliter, which showed 64.05% ± 0.45% progressive motility and 2.55% ± 0.75% DNA fragmentation [
5]. These differences may be due to the antioxidant paradox [
31] caused by GTE’s more potent antioxidant action than bull seminal plasma protein.
There were regression equations of all pre-freezing parameters, except the uncapacitated sperm to the post-thawed motility. The coefficient of determination (R2) is in the range of 19.25–97.30% (
p < 0.05), where the pre-freezing motility has the highest R2 to the post-thawed sperm motility (
Table 4 and
Figure 1). Therefore, frozen goat semen that qualifies for artificial insemination should reach at least 40% post-thawed sperm motility. Based on the regression equation of Pt Mot = −8.07232 + 1.07062 × Pf Mot (regression equation no.4,
Table 4), pre-freezing sperm motility that met the minimum requirements was obtained at 44.90%. It means post-thawed sperm motility was 89.08% of pre-freezing sperm motility.
4.3. Mutation of Amino Acids Encoded by NADH Dehydrogenase 1 (ND1) of Mitochondrial Deoxyribonucleic Acid (mtDNA)
This study is the first to report the amino acid mutations due to freeze–thawing. Based on semen quality parameters (sperm viability, motility, IPM, MDA levels, SDF, capacitation, and acrosome reaction), the T2 group had the highest quality. The frequency of amino acid mutations also revealed that T2 was the lowest. The results of this study were in accordance with our previous finding that the dose of 0.1 mg GTE/100 mL SM-EY extender is in the lowest nucleotide ND1 mtDNA mutation [
14]. It was shown that the addition of 0.1 mg GTE per 100 mL SM–EY extender might protect the lipid bilayers and membrane function [
32], decrease oxidative stress [
8], reduce DNA fragmentation [
35], and finally minimize the amino acid mutation.
Evolution theory revealed that maternal inheritance leads to the accumulation of mutations in mitochondrial DNA (mtDNA) that affect male fertility [
47]. Mitochondria have multiple functions, including the synthesis of adenine triphosphate, production of reactive oxygen species, calcium signaling, thermogenesis, and apoptosis. Mitochondria significantly regulate the various physiological aspects of reproductive function from spermatogenesis to fertilization [
48]. Each mitochondrion contains a covalently 16,569 bp DNA molecule that encodes 13 of the 83 subunits of the respiratory chain complex [
49].
A study of mtDNA mutation related to sperm preservation was found less often. In human sperm, we reported the difference in the ND1 mtDNA of asthenozoospermia compared to normozoospermic in Egyptian men. The ND1 gene in the asthenozoospermia sample yields ten detected SNPs, six of which are synonymous mutations in nucleotides T3396C, T3423C, C3594T, G3693A, G3705A, A4104G, and non-synonymous mutation in T3398C, T3821C, G4048A and insertion (T) 4169. Conversely, normal sperm samples produced four SNPs: two synonymous mutations at nucleotides A4104G and A4158G and two non-synonymous mutations in T4216C and insertion (T) 4169 [
50]. A higher prevalence of the mtDNA 4977 bp deletion was found in the subjects with impaired sperm motility and fertility. The mtDNA 4977 bp deletion, manganese superoxide dismutase (MnSOD, C47T), and 8-oxoguanine DNA glycosylase (hOGG1, C1245G) were accumulated in the sperm with poor motility [
51]. Disruptions of mitochondrial DNA (mtDNA) may affect male reproductive function in the Iranian population. The 4977 and 7599 bp deletions of mtDNA may be genetic risk factors for male infertility [
52]. Sixteen transition mutations have been predominantly detected withinside the Ghanaian populace’s mtDNA samples on the mutation of the amino acids. Missense mutations (threonine to alanine at positions 59, 112, 114, and 194 of the ATPase) present only in specific sperm abnormalities have been identified to contribute to male infertility [
49].
A higher sperm mitochondrial DNA copy number (mtDNAcn) and deletion rate (mtDNAdel) were correlated with semen parameters and were associated with lower fertilization [
53]. The mtDNA copy number was higher in asthenozoospermic semen samples and correlated negatively with sperm concentration, total sperm number, and motile spermatozoa. mtDNA content played a potential role as an indicator of semen quality. mtDNA copy number alterations and impaired chromatin integrity could affect reproductive success [
54]. The mtDNA copy number amount was higher in aneuploid embryos than in those euploids. However, there were no statistically significant differences in mtDNA content related to embryo morphology, sex, maternal age, or implant ability [
55].
4.4. The Correlation Coefficient of Amino Acid Mutation and Post-Thawed Kacang Buck Semen Quality Parameter
This study is the first to report the correlation of amino acid mutations to the semen quality parameters due to the freeze–thawing of animal semen. Three amino acid mutations (leucine, serine, and asparagine) strongly correlated with the post-thawed semen quality parameters. There was a significant correlation between a 7599 bp mtDNA deletion and asthenozoospermia in infertile Jordanian men. Furthermore, there was a significant correlation between 7599 bp mtDNA deletion and infertility, i.e., 63.6% in the infertile group compared to 34.2% in the fertile group. Additionally, the percentage of sperm motility showed a significant correlation to the frequency of that 7599 bp deletion [
56]. There was a correlation between the Single-Nucleotide Polymorphisms (SNPs) and male infertility in a Saudi population [
57].
Sperm fertilization capacities, including acrosin activity, acrosome reaction capability, and chromatin integrity, are related to mitochondrial functionality. Mitochondrial functionality might be necessary to maintain sperm acrosin activity, the acrosome reaction, and chromatin integrity [
58]. The potential membrane of mitochondri is a pre-requisite for sperm motility, hyperactivation, capacitation, acrosin activity, the acrosome reaction, and DNA integrity. Therefore, the optimum mitochondrial activity is crucial for sperm function and semen quality. Defects of sperm mitochondrial function cause disturbance to the energy production required for sperm motility. Sperm mtDNA is sensitive to the oxidative damage and mutations that affect sperm function and lead to infertility. Abnormal semen parameters have a higher mtDNA copy number and reduced mtDNA integrity [
48].
This study limited the extraction of green tea using ethanol solvent and the equilibration period to obtain semen quality that qualifies for artificial insemination. The one-hour equilibration period showed a satisfactory result. Meanwhile, the use of GTE antioxidants should be explored further. Among the different formulations, the nanoparticles are most effective in improving penetration into the cell [
59] and have a higher surface area to volume ratio [
60]. Future studies suggested using GTE nanoparticles on post-thawed semen quality, measuring the total antioxidant capacity and artificial insemination fields implementation. Since the post-thawed sperm motility is the crucial parameter of semen quality, attention needs to be focused on the glycine, valine, leucine, serine, and asparagine correlate strongly with post-thawed sperm motility. Sperm amino acid mutations due to SDF negatively impact pregnancy outcomes [
61]. Our ongoing study reveals that SDF produces offspring with low fertility.