The fresh goat semen samples in this study had a cream color, an average volume of 1.10±0.87 ml/time, a density of 4D (thick), a viscosity of 4
oC (milky), a pH of 6.53±0.14, a concentration of 17,203±275.18x10
6/ml and a motility of ++++ (75.7±0.13). The obtained fresh semen exhibited excellent characteristics, enabling the production of frozen semen with normal features such as a semen volume of 1-2 ml/time (
Settergren, 1934), a motility of at least ++ or 30 and a progressive motility rate of more than 70. The osmotic values of Tris-egg yolk (1,611 Osmol/kg) and MJ Ex2™ (1,708 Osmol/kg) for both semen diluent formulas were measured using an Osmomat 3,000 basic freezing point osmometer, Go Notes brand and they were found to be normal when
Nunes and Salgueiro’s (2011) freezing method was employed (Table 1).
The results indicated that the motility percentage of MJ Ex2™ extender was 76.72±5.00, which was a lot higher than the motility percentage of Tris-egg Yolk extender (66.48±2.89) (P<0.01). However, the percentages of forward motility, Path velocity (VAP), Straight-line velocity (VSL), Lateral head displacement (ALH) and Beat cross frequency (BCF) did not change. The Tris-egg Yolk extender formula’s motility was much higher, with a Linearity (LIN) value of 50.19±4.05 (P<0.01). The motility and Straightness (STR) values of the diluted sperm treated with MJ Ex2™ extender were much higher (78.14±5.04) than with the Tris-egg Yolk extender formula (72.60±7.93, P<0.05). When considering the sperm motility measurements, the research findings indicated that the MJ Ex2™ extender formula significantly enhanced the quality of frozen goat semen (Table 2), (Fig 1,2). In both groups of the experiment, sperm motility patterns showed that frozen goat semen mixed with MJ Ex2™ extender semen diluent contained more
viable than Tris-egg yolk formula. When statistically analyzed, a significant statistical difference was observed. It is possible that the use of the MJ Ex2™ extender formula in frozen goat semen made a significant impact by increasing certain important components such as gallic acid, ellagic acid and tannin acid. This outcome is consistent with the study of
Bo et al., (2010) who reported that gallic acid, ellagic acid and tannin acid could affect cells, all of which were related to the inhibition of cell growth and cell death and reduced free radicals. Furthermore, various semen diluent formulas for refrigeration or freezing, such as the Tris-fructose-egg yolk (TES-Tris) formula that uses fructose (
Foote, 1964) and the Tris-egg yolk formula, have been reported to incorporate different sugars as key components. Using glucose
(Province et al., 1984) or lactose
(Yubi et al., 1987) as reactive molecules helps to resist or prevent oxidation, thereby effectively maintaining the condition of cells and membranes. Moreover, studies have shown that the MJ Ex2™ extender semen diluent, which does not separate the seminal fluid and sperm before the sperm is diluted, might not be harmful or may not stop the substances in Cowper’s gland from reacting. Importantly, the production of frozen semen contradicts the findings of
Nunes et al., (1982) and
Sawyer and Brown, (1995) in cattle. In sheep, up to 20 egg yolk can be used
(Tometo et al., 2010; Roof et al., 2012). This can protect sperm from damage during the freezing process, reduce temperature and provide energy for sperm to move and survive
(Vidal et al., 2013). Mobility indicates the number of
viable sperm that are still able to move and are ready to travel in the female reproductive tract to the point of fertilization with an egg cell. The sperm can then complete all the processes of fertilization with the egg cell. The evaluation of the quality of semen after freezing would indicate that it is a good diluent.
The recipe for MJ Ex2™ extender goat semen cryotherapy is unique. The primary components are gallic acid, ellagic acid and tannin acid. This would align with the findings of
Bo et al., (2010), who found that gallic acid, ellagic acid and tannic acid all had a positive effect on cells by slowing down cell proliferation and reducing cell death. When goat semen is frozen or refrigerated, however, each formula has been found to use different sugars. For example, the Tris-fructose-egg-yolk formula uses fructose (
Foote, 1964), the Tris-egg-yolk formula uses glucose (
Province et al., 1984) and the egg-yolk formula uses lactose. These are reactive chemicals that help resist or inhibit oxidation, thereby protecting cells and membranes from freezing damage. This finding fits well with what
Naing et al., (2010) reported when they studied how the concentration of trehalose, a disaccharide sugar used in a Tris-containing semen freezing solution, affected their results. This research study demonstrated that monosaccharides, specifically glucose and fructose, significantly influenced sperm motility more than disaccharides at the examined amounts. The experimental results clearly showed that adding trehalose in different amounts, up to 198.4 mM with 69.38 mM glucose, had the most significant effect on the quality of the sperm after being frozen. Similarly,
Nunes and Salgueiro, (2011) found that glucose is a useful energy source for sperm and enhances sperm motility and direction. As a result, glucose is an ideal sugar for enhancing the goat semen freezing solution. However, the chemical makeup of goat sperm determines the impact of each form of sugar supplementation on sperm quality during the semen freezing procedure. Accordingly, electrolytes, amino acids, phospholipids and other related chemicals have been utilized including proteins and certain specific enzymes. This list would also include plasma, inacetal, glycerylphosphorylcholine and ergothioneine. Examining the specific composition of sperm obtained from different species did not necessarily reveal any differences
(Anel et al., 2003).
Furthermore, it was dependent upon the type of animal and the temperature at which the semen was stored, leading to two phases of semen quality storage, as follows: Short-term semen storage involves storing semen in liquid form (liquid semen) at a temperature of 5
oC for about one week with an acceptable fertilization rate. Long-term storage of semen quality involves storing it in a frozen state, which can be achieved in two ways: either packing it in straw tubes (ministraw) or freezing it at a temperature of -196
oC using liquid nitrogen to maintain the desired temperature. Another method would be to freeze the semen in pellets (pellets) using dry ice (solid carbon dioxide) to keep the temperature at -79
oC, which is not common at the moment because it can be cumbersome to thaw and involves combining diluted solutions and packing them in semen injection tubes. This liquid nitrogen cryopreservation method may keep sperm viable for an extended period of time; however, it has been observed that semen frozen for more than 40 years retains acceptable quality and has a high fertilization rate
(Anel et al., 2003). Apart from the diluent solution, there is another solution known as cryoprotectant that can effectively prevent the loss of semen quality. For instance, Polge Smith and Parkes discovered glycerol in 1948. Up until now, the production of frozen semen has widely used glycerol as a cryoprotectant. Other methods involve ethyleneglycol, dimethyl sulfoxide and propylene glycol. However, for bovine semen, glycerol works best to prevent the loss of cell properties during the freezing process. In this case, it acts physically rather than chemically
(Leboeuf et al., 2000). Additionally, each ejaculation’s properties can influence the semen, yielding distinctly different outcomes. Using the semen filtration method will help reduce the differences in semen quality caused by each ejaculation. This has been found to be important in ensuring consistent control standards for preservation and freezing (
Nunes and Salgueiro, 2011). To maintain the stability of the sperm cell membrane, many breeders of animal species use egg yolk or skim milk as a basic component in semen diluent to preserve semen quality. Lipoproteins and lecithin, or phosphatidylcholine, are found in egg yolks. In skim milk, casein protein is present, which is an important substance that helps to stabilize the cell membrane, reduce the loss of enzymes in the acrosomal membrane and prevent cell damage from temperature drops during both refrigeration and freezing processes. Notably, freezing semen in goats can encounter the same problems as the freezing of semen in other species. This is true of ice crystals forming in the cells during temperature reduction, which can damage the sperm cells. A good semen diluent must be able to maintain the condition of the sperm cells during the freezing process, prevent sperm cell damage from temperature changes and adjust the appropriate acid-based balance for sperm cells or buffers to make the resulting product as
viable as possible and close to natural semen (
Nunes and Salgueiro, 2011). It prevents microbial contamination from antibiotics being added to the semen diluent and is also an energy source for sperm cell movement (
Garner and Hafes, 1993). The semen diluent must be able to maintain the integrity of the cell wall and acrosome, while ensuring the forward movement of sperm cells, the strength of the sperm cells, the
viability of the sperm cells and the ability to provide a high fertilization rate
(Vidal et al., 2013).
The MJ Ex2™ extender formula revealed that sperm motility had higher STR values than Tris-egg yolk semen dilution, indicating that average sperm velocity and forward movement are crucial for sperm motility in the female reproductive canal. In terms of sperm motility, it has been determined that sperm with high motility are stronger and can go farther than sperm with the same motility speed. There is no information on which motility speed is best suited, yet sperm motility has clearly been linked to fertilization rate
(Correa et al., 1997). Research teams can expect the findings of this investigation to serve as a set of guidelines for future development and improvement in the production of frozen goat semen. We can conduct research by varying the number of ingredients until we achieve the desired amounts, or by incorporating additional compounds into the mixture. The cryoprotectant’s beneficial characteristics must allow sperm cells to survive and fully fertilize egg cells. It can also prevent damage to the cell membrane and acrosomes
(Nunes et al., 1982 and
Sawyer and Brown, 1995), lower the amount of harmful chemicals in cell metabolism or act as an antioxidant
(Zhang et al., 2022), stop cell shock after the temperature drops to -10
oC
(Ivan et al., 2022) and offer various other advantages. To evaluate the effectiveness of a semen diluent, we must dilute the semen based on the amount of sperm required for frozen goat semen production. When sperm cells remain in the semen diluent, it would indicate that the substance does not cause them to lose certain properties, which could lead to cell death or toxicity when mixed with other substances, thereby altering the surrounding environment and causing cell weakness. Researchers have been studying a limited number of cases where goats use semen diluent. Notably, Thailand’s data has become increasingly examined worldwide. However, the study and research of semen diluent continues to be an intriguing topic worthy of further pursuit, as it will benefit goat farmers throughout the nation. In terms of the application of knowledge, understanding goat artificial insemination enables farmers to employ more goat artificial insemination techniques, thereby assisting farmers in developing goat breeds and producing enough goats to fulfill global population demands.