Mangosteen peel extract in Tris-egg yolk extender improves fertility of cryopreserved goat sperm

Nurul Isnaini, Muhammad Nur Ihsan and Sri Wahjuningsih

Department of Animal Production, Faculty of Animal Science, University of Brawijaya, Malang 65145, Indonesia
nurulisna@ub.ac.id

Abstract

This study examined the effects of mangosteen peel extract (MPE) on cryopreservation and fertility of goat sperm. The semen was collected from a buck aged at 3 years old with the body weight of 60 kg. After the initial evaluation, the semen was then diluted in the Tris-egg yolk extender supplemented with MPE either at 0, 2, 4, or 6%. The results showed that sperm quality before freezing and post-thawing in all MPE treatments were decreased (p<0.05) compared to the fresh semen, but no significant difference (p>0.05) among MPE treatments on sperm quality before freezing and post-thawing. The use of 2% MPE tended to give optimum post-thawed semen quality traits. Post-thawed semen showed a positive correlation between sperm motility and viability, but they had a negative correlation with abnormality. The fertility test was conducted on 20 fertile does. The first group was inseminated with untreated frozen semen (control) (10 does), while other 10 does were inseminated with 2% MPE frozen semen. It was observed that 2% MPE resulting in a relatively higher non-return rate at 21 days than control (80% vs 70%). Overall, the inclusion of 2% mangosteen peel extract in Tris-egg yolk extender provide optimum goat semen characteristics post-thawing. The application of artificial insemination with frozen semen treated with 2% mangosteen peel extract could beneficially affect goat fertility.

Keywords: antioxidant, non-return rate, post-thaw, pregnancy rate, sperm characteristics

Introduction

Semen availability and quality are the crucial factors in artificial insemination (AI) program. Cryopreservation allows semen storage for a long period so that it could be available at all times. However, this process may induce oxidative damage, which leads to a decrease in sperm quality (Yimer et al 2014). The reduction of sperm quality will reduce its fertility (Layek et al 2016), which results in a failure of the AI program.

Recently, Tris-egg yolk extender is widely used in the semen cryopreservation (Ahmad et al 2018, Naz et al 2018, Swelum et al 2018). This extender has almost all nutrients required by sperm to maintain their quality, except for the antioxidant which is also a crucial compound to protect sperm from oxidative damage during cryopreservation. Adding antioxidant into Tris-egg yolk extender is required.

The mangosteen peel is accounted to about 65% of mangosteen fruit ( Garcinia mangostana L.) (Chaovanalikit et al 2012). Although considered as a by-product, this part has the desirable bioactive characteristics, particularly in terms of antioxidant activity. Previously, it was reported that mangosteen peel extract (MPE) contained 10,600 mg GAE/100 g and 29.7 mg cy-3-glu/100 g of total phenolic and anthocyanin contents (Chew and Lim 2018) as well as 40.8 to 49.8% of α-mangostin (Mulia et al 2018). In another study, Jaisupa et al (2018) discovered that the mangosteen peel has excellent antioxidant activity and may potentially act as cell protectors from oxidative damage. Recently, the use of mangosteen peel extract in the sperm cryopreservation has not been widely explored. Therefore, this study was carried out to assess the efficacy of the inclusion of mangosteen peel extract (MPE) in Tris-egg yolk extender on cryopreservation and fertility of goat sperm.

Materials and methods

Preparation of mangosteen peel extract and Tris-egg yolk extender

Mangosteen peel was mixed with aquabidest (1:10 w:v) and then ground using a food processor. The solution was then filtered using filter paper. After that, the filtrate was packed in the Eppendorf tube and stored in a freezer at -18^oC. Tris-egg yolk extender was prepared freshly every time by mixing 1.6 g Tris aminomethane (Merck, Germany), 0.9 g citric acid (Merck, Germany), 1.4 lactose (Sigma, USA), 80 ml aquadest, 20 ml egg yolk, 0.1 g penicillin (Sigma, USA), and 0.1 g streptomycin (Sigma, USA). The Tris-egg yolk extender was supplemented with either 0, 2, 4, or 6% MPE.

Experimental animals

The experimental animal used in this study was Senduro goat which was officially declared as one of the Indonesian native livestock genetic resources (Decree of Indonesian Minister of Agriculture Number 1055/Kpts/SR.120/10/2014). This goat was a result of the crossing among Indian Etawah goat x Kacang goat x Jawarandu goat which have been going on for 100 years.

Semen collection and dilution

Semen was collected from a Senduro buck aged at 3 years old with the body weight of 60 kg. The collection was done twice a week using artificial vagina method. After collection, the semen quality was immediately evaluated and the semen with individual motility of not less than 70% were selected for cryopreservation. After that, the semen was diluted in the extender with the ratio of 1:10 (v:v).

Assessment of semen quality traits

The semen quality traits were monitored before freezing and post-thawing. The sperm motility was assessed under a light microscope (400x magnification). The sperm viability and abnormality were examined using an eosin-nigrosin staining procedure (Reddy et al 2018).

Measurement of semen fertility

A total of 20 Senduro does aged between 2.5 and 3.5 years with the body weight ranged from 25 to 35 kg were used in this study. The does were healthy, had a normal reproduction cycle, at least had already kidding one time before the experiment, not pregnant yet, and at least at two months post-partum. To generate estrous synchronization, the does were injected intramuscularly with a single dose of PGF2α (Lutalyse^TM) (Zoetis, Ireland). The dose of PGF2α injection was 1 ml for those which had body weight of <30 kg and 1.5 ml for those which had body weight of ≥30 kg. The does were then divided into two groups (10 does of each group) and inseminated either with untreated frozen semen or those treated with the best inclusion level of MPE. The AI was conducted at 12 hours after estrous signs were observed. The straw (75 x 10⁶ sperm/straw) was firstly thawed and then inserted into the insemination gun (IMV Technologies, France). After that, the speculum (IMV Technologies, France) was inserted into the vagina of the does and finally the semen was deposited at the position of 1 to 1.5 cm intra-cervix. The estrous signs were again observed on each doe at 21±3 days after AI to anticipate the earlier or late estrous. The non-return rate at 21 days (NRR21) was calculated by using formula = (number of the doe with no estrous signs at 21 days after AI / total number of the doe received AI) x 100%.

Statistical analysis

All statistical procedures were performed using SPSS software version 13.0. The data on semen quality traits were analyzed using analysis of variance with LSD Duncan test to determine differences among the treatments. Statistical significance was set at p<0.05. The Pearson correlation test was applied to examine the relationship between semen quality traits post-thawing. The NRR21 was analyzed descriptively.

Results and discussion

Semen quality before freezing

Table 1 shows the characteristics of fresh semen in comparison with those before freezing as affected by the MPE treatments. It was clearly observed that the sperm motility and abnormality before freezing in all MPE treatments was decreased compared to the fresh semen. Moreover, the sperm viability before freezing in 0% and 2% MPE were statistically similar, while in 4% and 6% MPE were significantly lower than fresh semen. No significant difference was recorded among MPE treatments.

In this study, the use of MPE did not significantly affect sperm motility, viability, and abnormality before freezing. The mean sperm motility, viability, and abnormality were 56.88%, 69.73%, and 14.68%, respectively. Previously, Banday et al (2017) observed that the sperm motility and viability pre-freeze were not influenced by the supplementation of several antioxidant sources (taurine, quercetin, and reduced glutathione) in the extender. In another study, Khalifa and El-Saidy (2006) also reported that the antioxidant did not affect sperm motility post-dilution.

Semen quality post-thawing

This current study clearly showed that the cryopreservation could reduce the semen quality compared to the fresh semen (Table 2). In addition, it was found that among the MPE treatments, the highest sperm motility was recorded in 2% MPE, while the lowest one was recorded in 4% MPE. Although there was no significant difference among MPE treatment, 2% MPE exhibited the most preferred sperm viability and abnormality.

This current study showed that the cryopreservation of buck semen resulting in a decline of sperm characteristics traits. In agreement with this finding, Yimer et al (2014) also found that the post-thawed sperm motility and viability were lower, while the sperm abnormality was higher than the fresh semen. Other study by Gangwar et al (2015), found sperm motility reduces by 30.51 to 43.01% and viability reduces by 21.50 to 46.63 in post-thawed semen compared to the fresh semen The cryopreservation could induce cold shock, leading to the damage of sperm membrane so that the motile and viable sperm cell will be reduced (Morrell et al 2017).

The most preferred dose of MPE addition is 2% which was indicated by relatively better sperm motility, viability, and abnormality than the other treatments. In the previous study, the improvement of post-thawed semen quality was also observed with the inclusion of antioxidant sources such as avocado seed extract (Olamitibo et al 2016), bean sprout extract (Sumarmin et al 2018), Nigella sativa oil and honey (Maidin et al 2018). The ability of MPE to maintain sperm quality after thawing may be related to their antioxidant activity. Jaisupa et al (2018) showed that the mangosteen peel extract could exhibit antioxidant effect through scavenging reactive oxygen species and improve the release of antioxidant enzymes. However, it should be noted that the optimum antioxidant effects may be in a dose-dependent manner. As previously described by Eriani et al (2018), the antioxidant should be used in the appropriate dose to maintain the sperm quality during cryopreservation because the use of antioxidant at a too high level could induce a toxic effect.

Relationship among semen quality post-thawing

Table 3 shows the correlation between post-thawed sperm characteristics. The sperm motility had a positive correlation with the sperm viability, but they had a negative correlation with the sperm abnormality. The correlation among sperm characteristic in this study was similar to the previous findings. Chella et al (2017) also observed that there was a positive correlation between sperm motility and viability. In another finding, Daramola and Adekunle (2017) also reported a negative correlation between motility and abnormality.

Sperm fertility

The 2% MPE was chosen due to the relatively greater post-thawed sperm traits compared to the other inclusion levels of MPE. Figure 1 shows that 2% MPE in the extender has a higher NRR21 compare to non-added. The results showed that 7 out of 10 does did not show any estrous signs at 21 days after AI with untreated frozen semen. On the other hand, 8 out of 10 does did not show any estrous signs at 21 days after AI with 2% MPE frozen semen. Therefore, 2% MPE treatment resulting in the 10% higher NRR21 compared to 0% MPE treatment.

Figure 1. Non-return rate of does at 21 days (NRR21)

In this study, the addition of 2% mangosteen peel extract in Tris-egg yolk extender provides better fertility in term of NRR21 than the control treatment. The use of extender which contained lycopene and α-lipoic acid could increase pregnancy rate in Cashmere goat (Ren et al 2018). It could be expected that the antioxidant from mangosteen peel extract could protect frozen semen and resulting in higher fertility. The sperm motility and viability had a positive correlation with fertility (Januskauskas et al 2003). Santolaria et al (2015) also reported that sperm viability showed a significant impact on fertility.

Conclusions

• This study suggests that the addition of 2% mangosteen peel extract in Tris-egg yolk extender has more favorable post-thawed semen characteristics than the other treatments.
  
  
• The application of artificial insemination with frozen semen treated with 2% mangosteen peel extract could provide a beneficial effect on goat fertility.

Acknowledgment

The authors acknowledged financial support from Indonesian Ministry of Research, Technology, and Higher Education.

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Received 5 March 2019; Accepted 12 March 2019; Published 1 April 2019

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