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Supplementation of Moringa oleifera leaves’ extract in Tris-egg yolk extender on the quality and fertility of cryopreserved Senduro goat sperm

Sri Wahjuningsih, Gatot Ciptadi, Muhammad Nur Ihsan, Nurul Isnaini and Sri Rahayu1

Department of Animal Production, Faculty of Animal Science, University of Brawijaya, Malang 65145, Indonesia
1 Department of Biology, Faculty of Mathematics and Natural Science, University of Brawijaya, Malang 65145, Indonesia


The objective of this research was to analyze the effect of Moringa oleifera leaves extract (MLE) supplementation on the quality and fertility of Senduro bucks’ frozen semen. The semen was collected using an artificial vagina twice a week from a three-year-old Senduro buck with a body weight of 47 kg. The minimum standard of fresh semen is above 70% in individual motility and less than 10% in abnormality. Semen was diluted in Tris-egg yolk extender and boosted with MLE at 0, 1, 3 or 5% respectively. The observation revealed that sperm’s motility and viability, as well as plasm membrane’s integrity before freezing and post-thawing in all MLE treatments were lower (p<0.05) compared to those of the fresh semen. However, the use of 3% MLE resulted in the highest post-thawing semen quality traits. The test of sperm fertility was conducted on 50 fertile does. The does were divided into two groups. The first group (25 does) was treated as control (0% MLE), whilst the rest (25 does) were inseminated with 3% MLE. It is evident that 3% MLE had resulted in a relatively higher non-return rate (NRR) at 21-23 days than control (76% vs 56%). Hence, the supplementation of 3% MLE in Tris-egg yolk extender is able to improve the quality and fertility of the Senduro buck’s frozen semen.

Keywords: antioxidant, artificial insemination, frozen semen, sperm quality


Goat has been considered as one of the most popular livestock in Indonesian rural communities. Senduro goat a cross-bred from Ettawah and local goats, which has been adapting well to the Indonesian climate and environment. However, it is arguable that the reproductive management of goats is still far from sophistication, let alone professional management. One of the many examples of reproductive management is the preservation of the genetic potential of superior males by applying artificial insemination. In addition, artificial insemination is also beneficial to improve the efficiency of semen usage as semen from superior males can be utilized to fertilize more than one female (Dorado et al 2010; Gangwar et al 2016).

Semen availability and quality are important things in an artificial insemination program. Nearly half of the spermatozoa die during the cryopreservation process and the surviving ones generally possess low fertility level. Antioxidants need to be added to minimize the decline in the quality of semen due to cooling and freezing (Mishra et al 2010). Antioxidants are one of the compounds that are able to inhibit a decrease in semen quality due to processing by suppressing free radical reactions (Banday et al 2017). Hence, the quest to explore sources of high quality antioxidants has become essential.

In regards to that, it is reckoned that Moringa oleifera is a species of plant, which possesses high anti-oxidant contents, especially in its leaves (Das et al 2012; Verma et al 2009). Moringa leaves contain protein, fat, calcium, magnesium, potassium, selenium, vitamin C, vitamin E, and polyphenol compounds (Ayodele et al 2014), which are important to the survival of spermatozoa. Furthermore, phenolic compounds such as flavonoids, quercetin, and kaempferol in Moringa leaves are effective to enhance antioxidant activities (Moyo et al 2011). However, the use of Moringa leaves’ extract (MLE) in semen extender has not been widely explored. The previous study by Sokunbi et al (2015) showed the antibiotic potential of crude MLE in Friesian bull semen extender, while research by Fafo et al (2016) showed that 5% MLE concentration in citrate egg-yolk diluent had the best Landrace pig semen preservation. In this research, we further evaluate the supplementation of MLE in Tris-egg yolk extender on post-thawing quality and fertility of Senduro goat sperm. The result of this research is expected to provide alternatives for rural goat farmers to maintain their frozen semen qualities through semen extender supplementation with Moringa leaves’ extract, a relatively abundant plant in a tropical area such as Indonesia.

Materials and methods

Semen collection and dilution

Semen was collected from a three-year-old Senduro buck with a body weight of 47 kg twice a week using an artificial vagina. After collection, the semen’s quality was evaluated for standart individual motility of at least 70% and abnormality of maximum of 10%. Semen was then diluted in the extender with a proportion of 1:10 (v/v).

Preparation of Moringa leaves’ extract and Tris-egg yolk extender

Moringa leaves’ extract (MLE) was obtained based on methods employed in previous research (Sokunbi et al 2015). MLE was poured in 1.5 mL Eppendorf tube then kept in a freezer at -20°C. Tris-egg yolk extender was formulated by putting together 1.36 g Tris aminomethane (Merck), 0.76 g citric acid (Merck), 1.50 g lactose (Sigma), 0.50 g fructose (Sigma), 80 mL aquadest, 20 ml egg yolk, 0.1 g penicillin (Sigma) and 0.1 g streptomycin (Sigma). The Tris-egg yolk extenders were then divided into four groups, which were 10 ml Tris-egg yolk extender without MLE extender (control), 10 ml Tris egg yolk extender + 0.1 ml MLE (1% MLE),10 ml Tris egg yolk extender + 0.3 ml MLE (3% MLE) and 10 ml Tris egg yolk extender + 0.5 ml MLE (5% MLE).

Evaluation of semen’s quality

Sperm’s motility, viability, abnormality, and plasm membrane integrity were evaluated both before freezing and post-thawing. Sperm’s motility was examined under a light microscope with 400x magnification based on the percentage of progressive sperm’s motility (Lukman et al 2014). In addition, the evaluation of viability and abnormality was performed by conducting eosin nigrosin staining (Ducha et al 2012), whilst the observation of plasm membrane integrity was carried out by performing hypo osmotic swelling test (Amorim et al 2009).

Artificial insemination procedure

Fifty does aged 2.5 to 3.5 years with a body weight between 35 to 37 kg were used. The 50 sampled does all had a normal reproduction cycle, have had already kidding at least once, not pregnant yet, and ≥ 2 months post-partum. The does were equally divided into two groups (25 does each). The first group of does was inseminated with Tris-egg yolk with 0% MLE supplementation or considered as a control group, whilst the rest of 25 does (second group) was inseminated with the best frozen semen treatment from semen quality evaluation. All sampled female goats were injected subcutaneously with 1.5 mL of PGF2α (Lutalyse). The artificial insemination was executed 12 hours subsequent to the appearance of estrous signs.

All straws (75 x 106 sperm/straw) used in the experiment were thawed in warm water at 37oC for 30 seconds and then loaded into the insemination gun (IMV Technologies). The speculum was smeared with K-Y Jell and inserted into the vagina. After that, the semen was placed at the position of 1 to 1.5 cm intra-cervix using the artificial insemination gun. Subsequently, the estrous signs of each goat were observed at 21 to 23 days. Finally, the non-return rate at 21-23 days (NRR21-23) subsequent to the artificial insemination was calculated by using the formula = (number of goats with no estrous signs at 21-23 days after artificial insemination / total number of goats receiving artificial insemination) x 100%.

Statistical analysis

All statistical procedures were carried out using SPSS software version 21.0. An analysis of variance with LSD Duncan test was employed to discover the distinction among four treatments (0%, 1%, 3%, and 5% of MLE supplementation). Statistical significance was set at p<0.05 and the NRR21-23 data were descriptively analyzed.

Results and discussion

Semen quality before freezing

The semen quality of different concentrations of MLE before freezing can be seen in Table 1. The overall sperm motility, viability and abnormality of goat semen in this study is relatively similar to the finding by Isnaini et al (2019) which used mangosteen peel extract and Tris-egg yolk as frozen goat semen extender. Furthermore, supplementation of 3% MLE had shown remarkably higher motility, viability and plasm membrane integrity than other treatments (0%, 1%, and 5% MLE).

Table 1. Effects of Moringa Leaves’ Extract (MLE) supplementation in Tris-egg yolk extender on semen quality before freezing
Variable (%) Number of
Before Freezing SEM p value
0% MLE 1% MLE 3% MLE 5% MLE
Sperm motility 24 52.5b 51.5b 63.0c 49.0a 0.96 <0.001
Sperm viability 24 64.6ab 67.0b 75.8c 63.1a 0.54 <0.001
Sperm abnormality 24 5.29a 5.69b 5.19a 5.79b 0.12 <0.001
Plasm membrane integrity 24 66.8a 70.5b 77.5c 69.6a 0.71 <0.001
a, b, c ,d different superscripts within rows indicate significant differences at p<0.05
Semen quality post-thawing

It is evident that the motility, viability, abnormality, and plasm membrane integrity of Senduro buck semen post-thawing in all of MLE concentrations were lower than those of the fresh semen. As additional information, quality evaluation of fresh Senduro buck’s semen in this research showed the sperm motility, viability, abnormality and plasm membrane integrity at 80.5 %, 88.5 %, 4.49 % and 88.2 % respectively (excluded from the statistical analysis).

Table 2. Effects of Moringa Leaves’ Extract (MLE) supplementation in Tris-egg yolk extender on semen quality post-thawing
Variable (%) Number of
Post Thawing SEM p  
0 % 1% 3 % 5%
Sperm motility 24 46.5b 43.0a 51.5c 43.0a 0.71 <0.001
Sperm viability 24 45.6a 45.7a 55.4b 45.3a 0.78 <0.001
Sperm abnormality 24 6.02b 6.04b 5.19a 6.15c 0.13 <0.001
Plasm membrane integrity 24 60.5a 61.8b 73.4d 67.1c 0.52 <0.001
a, b, c ,d different superscripts within rows indicate significant differences at p<0.05

Previous research indicated that changes in temperature will trigger cells to experience physical and chemical destabilizations. Destabilizations increase cell membrane’s permeability to extracellular ions, including to calcium ions, which will result in an increase of intracellular calcium ions followed by an increase in calcium ions in the mitochondria (Ax et al 2000). Previous research had also found that the motility and viability of post-thawing semen were lower than those of the fresh semen (Isnaini et al 2019).

The supplementation of 3 % MLE resulted in a higher percentage of sperm motility, viability, and plasm membrane integrity of the treated group compared to control (Tris-egg yolk without MLE supplementation). The phenomenon can be explained by the presence of phenolic compounds containing antioxidants in MLE.

Antioxidant compounds will prevent the occurrence of lipid oxidation reactions in the spermatozoa’s plasm membrane (Nayak et al 2015; Banday et al 2017). This will enable spermatozoa to maintain their normal cell activity by maintaining pressure in the inside and outside parts of the cell and trigger the tail of the spermatozoa to circle upon encounter with hypoosmotic solutions. Previous research suggested that the supplementation of MLE could remarkably increase super-oxide dismutase and catalase, which at the same time led to a decrease in lipid peroxidation, thus significantly increase the quality of semen (El-Desoky et al 2017). The active components of MLE bind free radicals, thus offering protection against oxidative damages to macro cellular molecules. These free radical deterrents include polyphenols, flavonoids, and phenolic compounds (Chatterjee et al 2017). However, this study concluded that 5% MLE supplementation resulted in a lower quality of semen than those of 3% MLE. This finding is in line with the previous study, which argued that an excessive concentration of antioxidants may lose its effectiveness, in some cases, they even turn into prooxidants (Wahjuningsih et al 2019).

Correlation between semen qualities at post-thawing

In this study, we also observe the correlation between semen qualities (sperm motility, viability, abnormality and plasm membrane integrity) from 24 frozen Senduro buck’s semen during post-thawing, thus a total of 96 correlations were observed. Table 3 shows the correlation between semen qualities at post-thawing. It is noted that motility had a positive correlation with viability and plasm membrane integrity, whilst negative correlation is evident between motility and abnormality. The result is in line with those observed on the previous studies where there was a positive correlation between motility and viability as well as motility and plasm membrane integrity (Chella et al 2017, Lodhi et al 2008, Matabane et al 2017). Whereas in another study, Sharma et al (2012) reported a negative correlation between motility and abnormality.

Table 3. Correlation between semen qualities at post-thawing
Plasm membrane
Sperm motility 1 0.947** -0.663** 0.877**
Sperm viability 0.947** 1 -0.656** 0.928**
Sperm abnormality -0.663** -0.656** 1 -0.674**
Plasm membrane integrity 0.877** 0.928** -0.674** 1
**Correlations are significant at p<0.01
Implementation of artificial insemination

It was found that among MLE treatments, the highest quality of semen at post-thawing was recorded in the Tris-egg yolk with 3% MLE, hence was chosen for the artificial insemination. It is evident that the 3% MLE concentration in Tris-egg yolk extender had been able to provide a higher NRR21-23 than those of Tris-egg yolk without MLE supplementation. The research found that 14 out of 25 does (56%) showed no estrous sign on 21-23 days subsequent to the artificial insemination using Tris-egg yolk without MLE supplementation. On the other hand, 19 out of 25 (76%) does show no estrous signs on 21-23 days subsequent to the artificial insemination using 3% MLE frozen semen.

Therefore, the application of artificial insemination using 3% MLE supplementation in Tris-egg yolk extender had resulted in 20% higher NRR21-23 than 0% MLE. In other words, the supplementation of 3% MLE in Tris-egg yolk dilution may potentially provide better fertility rate in terms of NRR21-23 days than control (Tris-egg yolk with 0% MLE supplementation). In addition, the sperm’s motility, viability, and plasm membrane integrity in Tris-egg yolk extender with 3% MLE supplementation were higher than control. The result is in line with previous studies, which had shown that there was a positive correlation between motility and fertility (Januskauskas et al 2003, Santolaria et al 2015). Hence, it can be said that the supplementation of 3% MLE in Tris-egg yolk extender could increase frozen semen’s quality and fertility.



The research is supported by a grant from Indonesia government year 2018 (Contract Number: 012/SP2H/PPM/DPRM/2018 and 054/SP2H/LT/DPRM/2018). Authors would like to express sincere gratitude to goat farmers of Toyomarto, Singosari village in Malang Regency for the facilities provided, especially to Mr Rochim for the technical assistance.


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Received 10 August 2019; Accepted 31 October 2019; Published 2 December 2019

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