The Quality of Boer Goat Semen Preserved with Sugar Palm Juice

The objective of this study was to examine the effect of seminal plasma on viability of Boer goat spermatozoa and effectiveness of sugar palm juice as an alternative extender during preservation at 5oC. Semen of two Boer goats were collected using an artificial vagina. Fresh semen were evaluated and divided in equal volume into four tubes. Semen in the first and second tubes diluted with 80% sugar palm juice + 20% egg yolk (P1) and Andromed (P2), respectively. Semen in the third and fourth tubes were centrifuged with 3,000 RPM for 20 minutes, and the supernatant removed. diluted with 80% sugar palm juice + 20% egg yolk (P3) and Andromed (P4), respectively. Dilutedsemen were preserved in refrigerator at 5C, and quality of the spermatozoa including motile spermatozoa (MS), live spermatozoa (LS), and intact plasma membrane (IPM) were evaluated every day for four days. Results of this study showed that at day-2 preservation, mean percentages of MS, LS, and IPM for P2 (72, 83.4, and 83.4%), P3 (72, 82.6, and 82.2%), and P4 (72, 83, 83.8%) were significantly (P<0.05) higher than P1 (3, 24.8, and 25.2%). At day-3 preservation, mean percentages of MS, LS, and IPM for P2 (57, 65.6, and 69.6%) was significantly (P<0.05) higher than P3 (21, 34.8, and 31.8%), P4 (22, 33.6, and 31.2%), and P1 (0, 0, and 0%). In conclusion, semen of Boer goat to be preserved with extender containing egg yolk should be removed seminal plasma. Sugar palm juice containing egg yolk could be used as an extender for Boer goat semen, but should be applied in the AI program immediately after the semen is diluted.


Introduction
Boer goat is a type of meat goats with a good body conformation, adaptable to changes in ambient temperature, and more resistant to disease (Malan, 2000).The Boer goat is a superior goat imported from South Africa.One of the objectives of importing Boer goats is to improve the performance of Indonesian local goats, such as Kacang goats.According to Inounu et al. (2002) one way to increase livestock productivity is to include superior males from foreign breeds to be crossbred with local females.
The application of artificial insemination (AI) technology is a method that can be carried out to accelerate the population growth and to improve the genetic quality of goats.Through the integration of AI into semen processing technology, the reproductive potential of superior males can be optimized for mating with females.This objective can be achieved if the semen is diluted with certain diluents, meeting the quality requirements such as being energy source, buffer capacity, non-toxic, preventing damage to spermatozoa, cheap, and easy to obtain.According to Rizal et al. (2003) one ejaculation of male sheep can be used to mated about 35 females when using an AI program by using frozen semen packed in a mini straw; compared to the natural mating where one ejaculation can only be used to mated one female.
One of the main problems in handling goat semen is the presence of enzyme in seminal plasma.The enzyme is synthesized by the bulbouretralis glands (Cowper glands) which, when interacting with egg yolk or milk, will cause coagulation of semen (Ritar and Salamon, 1982;Leboeuf et al., 1998;Leboeuf et al., 2000).The enzyme is identified as phospholipase A that can hydrolyzed yolk lecithin into fatty acids and lysolecithin.These fatty acids and lysolecithin from the hydrolysis are toxic to goat spermatozoa.Meanwhile, in the process of semen processing, the egg yolk and milk are commonly used as the components of the semen extender.The lecithin contained in the egg yolk is the main reason for its use as the component of semen extender.Lecithin is needed as the protector of spermatozoa plasma membrane from cold shock when semen is stored at low temperatures (Quinn et al., 1980;Watson, 1981).This problem can be solved by two alternative methods.The first method: if one of the components of semen extender is egg yolk, so the seminal plasma should be removed by centrifugation.According to Ustuner et al. (2009) and Naing et al. (2011) the separation of seminal plasma increases the spermatozoa motility of Saanen and Boer goats after thawing.The second method: if seminal plasma is not removed, it is better to use a semen extender that does not contain egg yolk.Andromed is one of the soybean-based commercial semen extender that does not contain egg yolk, so it can be used for an alternative extender in preservation of goat semen.
The utilization of natural components such as sugar palm juice as the component of semen extender can be an alternative to synthetic chemical compounds which are expensive.Sugar palm juice contains 9.16% water, 84.31% sucrose, 0.11% fat, 2.28% protein, 3.66% total mineral, 1.35% calcium, and 1.37% phosphorus (P2O5) (BPTP Banten, 2005).Sugar palm juice also contains vitamin A and C and has a pH of 6-7.This fact about the content of chemical compounds is the basis why sugar palm juice can be utilized as one of the alternative semen extender.Sugar palm juice as an alternative extender has been reported to work well in the preservation and application of AI in swamp buffaloes (Rizal and Riyadhi, 2016).The objectives of this study was to examine the effect of seminal plasma on viability of Boer goat spermatozoa and the effectiveness of sugar palm juice as an alternative extender during preservation at 5 o C.

Collection, treatment, and preservation of semen
Semen was collected from two male adult Boer goats using artificial vagina.The fresh semen was evaluated for the quality including: volume, mass movement, concentration, degree of acidity (pH), percentage of motility, percentage of abnormality (morphology), percentage of live, and percentage of intact plasma membrane.The fresh semen that qualifies for AI (percentage of motility  70%, concentration ≥ 2,000 x 10 6 cell/ml, and abnormality < 15% (Bearden and Fuquay, 1997), is processed into liquid semen.
The fresh semen was divided into four tubes in equal volume.The semen in the first and second tubes were diluted with 80% sugar palm juice + 20% egg yolk (P1) and Andromed (P2), respectively.The semen in the third and fourth tubes were centrifuged at 3,000 RPM for 20 minutes and seminal plasma was removed.The pellets (spermatozoa) in the third and fourth tubes were diluted with 80% sugar palm juice + 20% egg yolk (P3) and Andromed (P4), respectively.The diluted-semen in the tubes inserted into a glass containing clean water and then preserved in refrigerator at 5ºC.The quality of spermatozoa were evaluated daily until the speramtozoa motility reached 40%.
Sugar palm juice to be used as the extender was first heated to boil without using additives.The heated sugar palm juice was then filtered with filter paper and stored at 5°C for 12 hours before being used as the semen extender.The Andromed extender was consisted of 20% Andromed and 80% aquabidest.All extenders were added of 1,000 μg penicillin and 1,000 IU streptomycin per milliliter extender.Semen was diluted to a concentration of 100 million motile spermatozoa per milliliter.

The variables of spermatozoa quality
The spermatozoa quality variables that were observed including: motility, live spermatozoa (viability), and spermatozoa that had intact plasma membranes.The motility of spermatozoa is the percentage of proportion of progressive moving spermatozoa (moving forward).The motility of spermatozoa was evaluated subjectively using a 400x magnification microscope in eight different fields (Rasul et al., 2001).
The viability of spermatozoa is the percentage of live spermatozoa, which is evaluated using eosin-nigrosin staining (Felipe-Perez et al., 2008).Spermatozoa with a white or transparent head are live spermatozoa, while the dead are marked by a red head.Spermatozoa were evaluated for a minimum of 200 using a 400x magnification microscope.
Abnormal spermatozoa are the percentage of spermatozoa with primary abnormalities, including abnormalities of the head and tail.Abnormal spermatozoa were evaluated using preparation used for evaluation of spermatozoa viability (Felipe-Perez et al., 2008).A minimum of 200 spermatozoa were evaluated with a 400x magnification microscope.
Intact plasma membrane is the percentage of spermatozoa that have intact plasma membrane.This variable was evaluated using the method of hypo-osmotic swelling (HOS) test (Fonseca et al., 2005).The hypo-osmotic solution consisted of: 1.351 g fructose + 0.735 g sodium citrate diluted with aquabidest until 100 ml volume.A total amount of 1.000 μl hypo-osmotic solution were added with 50 μl semen and mixed until homogeneous and then incubated at 37°C for 30 minutes.The thin layer preparation in the object glass was evaluated by a 400x magnification microscope to a minimum of 200 spermatozoa.Spermatozoa with intact plasma membrane are characterized by a circular or bulging tail, while the damaged are marked by a straight tail.

Data analysis
Data on motility, viability, and intact plasma membrane of spermatozoa were collected using a completely randomized design with four treatments and five replications, and the differences of between treatments were tested using the least significant different test.The data were processed using SPSS version 6.0 for windows (SYSTAT, 1996).
Based on the value of fresh semen quality, it can be concluded that the semen produced by Boer goats in this study qualifies to be processed into liquid semen or frozen semen.Good fresh semen should have a percentage of motile spermatozoa  70% and mass movement of ++ or +++ (Evans and Maxwell, 1987), a percentage of abnormal spermatozoa 6 -10% (Delgadillo, 1992;Ax et al., 2000), and a percentage of intact plasma membrane  60% (Revell and Mrode, 1994).

Quality of spermatozoa during semen preservation
The results showed that on the second day of preservation, the semen containing seminal plasma and diluted with egg yolk-containing extender had a very drastically reduced quality of spermatozoa.On the second day of preservation, the mean percentage of motile spermatozoa for P1 (3.8%) was significantly (P < 0.05) lower than P2 (72%), P3 (72%), and P4 (72%) (Table 2).The same was true of the percentage of live spermatozoa and intact plasma membrane (Table 3 and Table 4).
Based on the observation it was found that the Andromed extender (treatment P2) was the best treatment because it was able to maintain the quality of spermatozoa for three days of preservation.
Andromed contains various substances such as Tris hydroxy aminomethane, citric acid, fructose, lecithin, glycerol, and antibiotics (Minitüb, 2001) that are needed to protect spermatozoa during preservation at 5 -10 o C. Lecithin (phosphatidyl choline) contained in the Andromed extender is the product of extracts from soybeans.This soy lecithin has the same function as lecithin contained in egg yolks, which protects the spermatozoa from the damage caused by cold shock.According to Kayser et al. (1992) and White (1993), lecithin in egg yolks protects spermatozoa from bad effects of cold shock.
On the third day of preservation, the quality of semen diluted in the absence of seminal plasma (treatment P3 and P4) were lower than that of semen containing seminal plasma and diluted with Andromed (treatment P2) (Table 2, 3,  and 4).This phenomenon shows that seminal plasma has an important role in maintaining the life of spermatozoa.The seminal plasma contains various nutrients, which may play a role in protecting spermatozoa during preservation, cannot be completely replaced by the nutrients contained in the extenders.The seminal plasma contains various nutrients such as carbohydrates, proteins, vitamins, minerals, hormones, etc. that serve to support the life of spermatozoa (Hafez and Hafez, 2000).Rizal et al. (2008) reported that the semen of Etawa Crossbreed (EC) goat, which was diluted with egg yolk-containing extender without the removal of seminal plasma, was coagulated.This caused the spermatozoa to die.Afterwards, it was reported that the quality of EC goat spermatozoa preserved at low temperatures without seminal plasma was lower than that of spermatozoa preserved with ram seminal plasma.
The results obtained in this present study differ from those reported by some previous researchers.Alawiyah and Hartono (2006), Kostaman and Sutama (2006), Pamungkas and Anwar (2013), Rosmaidar et al. (2013), Suharyati and Hartono (2013), Pamungkas et al. (2014), andRhochim et al. (2017) reported that Boer goat semen diluted with egg yolk-containing extenders did not cause spermatozoa death when preserved at 5°C.This may be due to concentration differences of the phospholipase A enzyme in seminal plasma.
The results showed that the Boer goat semen diluted with Andromed and without removed seminal plasma (treatment P2) could be preserved at 5°C for three days and still feasible to be used in the AI program, because the percentage of motile spermatozoa was about 40%.Based on the Indonesian National Standard (SNI, 2014), the eligible goat semen used in the AI program must have a minimum percentage of motile spermatozoa of 40%.The semen diluted with treatment P3 and treatment P4 could be stored for one day, while the semen diluted with treatment P1 should be used in the AI program immediately after it was diluted.

Conclusion
It can be concluded that the Boer goat semen to be stored with extender containing egg yolk should be separated from the seminal plasma.Andromed is the best diluent for Boer goat semen, and can maintain quality of spermatozoa for three days of preservation at 5°C.Sugar palm juice containing egg yolk can be used as the Boer goat semen extender, but it should be applied in the AI program immediately after the semen is diluted.
Agriculture, Lambung Mangkurat University for the support and assistance so that this research can be finished.

Table 1 .
Mean characteristics of Boer goat fresh semen

Table 2 .
Mean percentage of motile spermatozoa during preservation at 5 o C

Table 3 .
Mean percentage of live spermatozoa during preservation at 5 o C