Objective: The aim of this study was to ascertain the effects of adding green tea extract (GTE) to skim milk-egg yolk (SM-EY) extender on both the quality of post-thawed bull semen and the pregnancy rates of the recipient cows.Methods: Twelve ejaculates from four Simmental bulls, aged 3 to 5 years and weighing 900 to 950 kg, were diluted SM-EY extender, added with 0, 0.05, 0.1, and 0.15 mg GTE/100 mL extender and then frozen. After four weeks storage in liquid nitrogen, the sperm were thawed and evaluated for viability, motility, intact plasma membrane (IPM), and DNA fragmentation. Meanwhile, the estrus cycles of 48 recipient cows were synchronized by intramuscular administration of a single injection of 5 mg prostaglandin F2α. Estrus cows were divided into four equal groups and inseminated artificially 18 to 20 h after the onset of estrus by using semen from each extender group. Pregnancy was diagnosed by measuring serum progesterone levels at 21 days, followed by transrectal palpation 90 days after insemination.Results: The findings revealed that adding 0.1 mg of GTE/100 mL extender produced the highest percentages of sperm viability (70.67%±1.75%), motility (69.17%±1.47%), and IPM (69.23%±1.21%) and the lowest percentage of DNA fragmentation (3.00%±0.50%). The pregnancy diagnosis revealed that all cows (36/36) inseminated using frozen semen in GTE addition extender were pregnant (pregnancy rate 100%), whereas the pregnancy rate of the control group was 83.33% (10/12).Conclusion: It may be concluded that 0.1 mg GTE/100 mL extender yields the best quality of spermatozoa and that all variants doses of GTE in extender produce a higher pregnancy rate among recipient cows.

The study aimed to determine the addition of green tea extract (GTE) in extender on the quality and DNA mutation of post-thawed Kacang buck sperm. The sperm DNA mutation was observed on nicotinamide adenine dinucleotide hydride (NADH) dehydrogenase 1 (ND1) of mitochondrial Deoxyribonucleic Acid (mtDNA). A pool of 12 Kacang buck ejaculates was diluted in skim milk-egg yolk extender contained 0, 0.05, 0.10, and 0.15 mg of GTE/100 mL for T0, T1, T2, and T3 group, respectively. Each of the aliquot groups was packaged in 0.25 mL French mini straw contained 60 million alive sperm and froze according to the protocol. The ND1 mtDNA amplification of samples was carried out Polymerase Chain Reaction machine, followed by DNA sequencing using the Sanger method. Meanwhile, the phylogenetic tree was constructed using the neighbor-joining (NJ) method with MEGA 7.0 software. The results showed that the T2 group maintained the highest quality for Kacang buck post-thawed semen. There was the highest percentages of sperms viability, motility, intact plasma membrane (IPM), the lowest of malondialdehyde (MDA) concentration, sperm DNA fragmentation (SDF), the total and types of ND1 mtDNA mutation frequency. The phylogenetic tree analysis revealed that the clade of the T2 group was most closely related to the sequence reference. However, there was no correlation between the semen quality parameters (sperm viability, motility, IPM, MDA concentration, and SDF) with ND1 mtDNA mutation of post-thawed Kacang buck semen. It could be concluded that GTE was useful as an antioxidant for Kacang buck semen extender for frozen sperm.

Aim: The purpose of this study was to determine the benefits of L-arginine addition in skim milk extender to maintain the quality of goat spermatozoa in chilled storage. Materials and Methods: A total of 18 ejaculates from three healthy goats with weight and age of 45 kg and 4-5 years, respectively, were divided into three groups. The control group contained goat semen diluted in a skim milk extender without L-arginine; Treatment I and Treatment II contained goat semen diluted in a skim milk extender with added L-arginine 4 and 6 mM, respectively. These three groups were chilled at 5°C and evaluated daily for 5 days. Observed variables were viability, motility, intact plasma membrane (IPM), malondialdehyde (MDA) level, necrosis, and apoptosis of spermatozoa. Results: The addition of L-arginine 4 mM was the best treatment in maintaining viability, motility, and IPM and a decreased MDA level, percentage of necrosis, and apoptosis of goat spermatozoa. An ejaculate in this extender can be divided into 37 doses for intracervical insemination in <1 ml volume with 125 million motile spermatozoa. Conclusion: Goat semen retained its quality when kept for 5 days in chilled storage by adding L-arginine in skim milk extender.

This study was the first to combine the addition of antioxidants to a skim milk–egg yolk (SM–EY) extender and different equilibration periods to obtain higher quality post-thawed Kacang buck semen. This study aimed to determine the effects of green tea extract (GTE) on the quality of frozen Kacang goat sperm equilibrated for one and two hours. The pool of Kacang buck ejaculate was equally divided into four portions and was diluted in an SM–EY extender that contained four doses of 0, 0.05, 0.10, and 0.15 mg of GTE/100 mL for T0, T1, T2, and T3 groups, respectively. The aliquots were treated for an equilibration period of 1–2 h before further processing as frozen semen. Post-thawed semen quality was evaluated for sperm quality. The Sanger method was used for DNA sequencing, and the amino acid sequence was read using MEGA v.7.0. The post-thawed semen of the T2 group that was equilibrated for one hour had the highest semen quality. Pre-freezing motility had the highest determination coefficient compared to post-thawed sperm motility. This study is the first to report amino acid mutation due to freeze–thawing. The frequency of amino acid mutations revealed that T2 was the least mutated amino acid. Glycine, valine, leucine, serine, and asparagine strongly correlated to post-thawed sperm motility. It can be concluded that a combination of 0.1 mg GTE/100 mL extender as an antioxidant and one-hour equilibration period resulted in the best post-thawed Kacang buck semen quality.