The Success Rate of Artificial Insemination Using Post-Thawed Spotted Buffaloes Epididymal Sperm

Spotted buffalo, an exotic species that exists in Tana Toraja, South Sulawesi, Indonesia, is getting extinct due to high number of slaughtered during a funeral ceremony, called Rambu Solo', as well as special treatments that do not allow the male spotted buffaloes perform natural mating activity. According to that, the research was trying to start conservation program by collected the cauda epididymal sperm soon after slaughtered. Two egg yolk-based extenders with different buffers, tris hydroxyl amino methane (TEY20) and trisodium citrate dehydrate (CEY20), were used as comparison to evaluate the post-thawed epididymal sperm quality and fertilizing capacity in artificial insemination program. The results showed that the post-thawed progressive motility of epididymal sperm was 40% and 39.17%, while viability was 65.99% and 63.26% and membrane integrity was 65.43% and 63.03% in TEY20 and CEY20 extenders, respectively. The success rate of pregnancy was 46.67% using post-thawed epididymal sperm in TEY20 and 40% using the one in CEY20 extenders. In conclusion, tris-based and citrate-based extenders have similar ability to maintain the epididymal sperm quality and its fertilizing capacity.


INTRODUCTION
Spotted buffalo (Bubalus bubalis carabanensis) is a sub-breed of Asiatic swamp type, which is belong to water buffalo classification. Male spotted buffaloes have strong bound with the culture and tradition in Tana Toraja, where this animal exists. Male spotted buffaloes have been used as prestigious symbol in social status that showed in funeral ceremony, called Rambu Solo'. In this tradition, hundred of animals, mainly male spotted buffaloes and including cattle, pig, horse, deer, chicken, etc., are slaughtered as an expression of sorrow and condolence. It is believed that the more male spotted buffaloes slaughtered the faster ride to reach paradise. Because of this myth and cultural value, male spotted buffaloes get special treatments while they are alive, any mating activities are not allowed. Consequently, the population is significantly decreased and the cost of a fully-grown male spotted buffaloes is 10 times higher than the normal ones, neither solid nor white bulls.
An overcoming problem solution is absolutely needed to protect the existence of male spotted buffaloes in the world. One possible technique that can be applied to conserve male spotted buffalo is artificial insemination (AI) using sperm from cauda epididymal tissues that collected from slaughtered male spotted buffalo during the funeral ceremony. It has been known in other species that cauda epididymis sperm has similar capability to fertilize the eggs and have resulted normal births (Lone et al., 2012). This technique has been considered for utilization of gamete from valuable animals or animals in risk of extinction (Kaabi et al., 2003). It is confirmed in our previous study that fresh male spotted buffalo's epididymal sperm has equal quality with the one from ejaculation (Yulnawati et al., 2010). That study was using commercial extender with some modification. Chemically defined is cheap, easy to prepare and fresh extender. It is more applicable to be used for spotted buffalo semen cryopreservation, because there are some limitations of the commercial extenders, i.e. expensive price with short period of using and limitation access for the product, since the location of male spotted buffaloes are far from the lab. In this study, our objective was to evaluate the post-thawed epididymal sperm quality in chemically defined extenders and its correlation to success rate of pregnancy and parturition rate in AI program.

Cryopreservation of Epididymal Sperm
Six pairs of cauda epididymis tissues from six to ten years old slaughtered male spotted buffaloes were collected during the funeral ceremonies in North Toraja, South Sulawesi. Scrota-coated of testes-epididymus tissues were transported in NaCl 0.9%, room temperature for 1-2 h. The cauda epididymal sperm was collected using both slicing and flushing methods in two different extenders, tris hydroxyl aminomethane-20% egg yolk (TEY20) or trisodium citrate dehydrate-20% egg yolk (CEY20). The composition of TEY20 and CEY20 extenders were referred to Singh et al. (2007) and Lone et al. (2011), respectively (Table 1). Fresh and post-thawed sperm quality evaluation, sperm dilution, equilibration, cryopreservation and thawing procedure were referred to Yulnawati et al. (2010).

Artificial Insemination
Thirty female spotted buffaloes were injected using double dosage of prostaglandin (PGF 2α ) with 11 d of interval to synchronize the estrous periods. The sperm used in this experiment was collected from the same bull to avoid bias of AI results because of male individual factor. Estrous detection was performed 72-84 h after second PGF 2α injection. Female spotted buffaloes were inseminated twice within 8-12 h intervals in between using single straw per insemination (concentration 60.10 6 sperm/straws). The pregnancy rate was confirmed by rectal palpation at day 90-120 after insemination. Success rate of AI was proved by number of offspring that delivered in AI program.

Data Analysis
All individuals were considered as the replications in these experiments. Results were expressed as the means ± standard error mean (SEM) and analyzed using t-test within 5% (P<0.05) level of confidence to determine statistical differences.

RESULTS AND DISCUSSION
Fresh sperm that obtained from cauda epididymal tissues of male spotted buffaloes has good quality according to the requirements for artificial insemination and also for the cryopreservation process (Indonesian National Standard, SNI 01-4869.2-1998). The excess using from cauda epididymis is high concentration of sperm that could be collected since it is the depository before ejaculation. As it has shown in our results, the obtain concentration of male spotted buffaloes epididymal sperm was high with good quality in motility and other parameters (Table 2). It is well known that the important the physiological characteristic differences of epididymal versus ejaculated spermatozoa is in the membranes composition that affect the survival rate of sperm cell after cooling and freezing (Martinez-Pastor et al., 2006). Reports in some species indicated that epididymal spermatozoa are less tolerant to cryopreservation process than ejaculate spermatozoa (Martins et al., 2009). Furthermore, epididymal spermatozoa has not been exposed to the complex secretions of the accessory sex glands that also important for during chilling and freezing process (Yu & Leibo, 2002).
There was no significant different (P>0.05) of sperm quality in all parameters, i.e. progressive motility, viability and membrane integrity during cryopreservation process (Table 3), as well as the success rate of pregnancy in TEY20 versus CEY20 groups (Figure 1). Both tris-and citrate-based extender seems to have equal maintenance ability as well as fertilizing capacity that was described on the pregnancy and delivery rate following artificial insemination program.
The decreasing temperature that happened extremely during cryopreservation process induces the formation of intracellular ice crystals and the osmotic and chilling injury of sperm cells (Isachenko, 2003). Ice crystal formation caused sperm cells injured and being damage to the cytoplasmic fracture; negative effects on the cytoskeleton and genome related structures; negative effect on sperm tail and its motile ability as well as reducing the fertility due to compromising the integrity of plasma membrane (Harshan et al., 2005;Andrabi et al., 2008). Cold shock influences the ratio of cholesterol/phospholipids, the content of lipids in the bilayer membrane, degree of hydrocarbon chain saturation and ratio of protein/phospholipid on the plasma membrane (Medeiros et al., 2002), reduces membrane permeability to water and solutes and injures acrosomal membranes (Purdy 2006), as well as mitochondrial sheath and axoneme (Salamon & Maxwell, 2000). A good extender is a combination of compositions that maintains the pH balance to protect membrane intactness from ice crystal damage and suitable for cell nutrition needs during cryopreservation process and storage. Membrane integrity is important for motility and viability of cells and also as indicator of fertilizing ability (Mehmood et al., 2009).
Tris-egg yolk extender has been widely recommended for routine use to cryopreserve male buffalo semen. The progressive motility of post-thawed ejaculated sperm of Nili-Ravi buffalo in tris-based extender was 48.8% (Waheed et al., 2012;Akhter et al., 2011a), while in post-thawed epididymal sperm of African buffalo (Synceraus caffer) were 56.4% in Triladyl and 44.1% in AndroMed (Herold et al., 2004). It is known that trisand milk-based extender could maintain sperm quality better that citrate-based extender during cryopreservation (reviewed by Andrabi 2009;Sansone et al., 2000). Nevertheless, we still believe that the citrate-based extender capacity could be improved by some modification of its chemicals composition. The advantages of citrate-based extender were found sufficiently clear for motility assessment compare to commercially milkbased extender (reviewed by Sansone et al., 2000).
Several reports of previous studies showed that sugar addition into extender was beneficial as extracellular cryoprotectant, as well as energy source for the sperm itself. As a non-permeable cryoprotectant, sugar  1. The success rate of pregnancy from artificial insemination program using spotted buffalo frozen-thawed epididynal sperm; TEY20 (■): tris hydroxyl amino methane-20% egg yolk; CEY20 (□): trisodium citratedehydrate-20% egg yolk. induces the increasing of osmotic pressure that could let the cell dehydration and reduce the incidence of intracellular ice crystal formation (reviewed by Andrabi 2009). Based on this information, fructose was added into citrate-based, as well as tris-based extender and it is shown in this study that both extenders could maintain post-thawed epididymal sperm quality and produce equal success rate of pregnancy (Table 3 and Figure 1). Parallel to our result, egg yolk-citrate-fructose extender also has similar potential in maintaining the viability and acrosomal integrity of ovine epididymal sperm during preservation at 4 ºC for 72 h (Lone et al., 2012). Compared to post-thawed ejaculates sperm quality of Nili-Ravi buffaloes (Akhter et al., 2010;2011a), the quality and fertility of post-thawed epididymal sperm of spotted buffalo was not so much different. The delivery rate was 46.67% in TEY20 versus 40% in CEY20 in this study. The conception rate in river buffalo using post-thawed ejaculate sperm in different extenders was reported in widely range, i.e 44% in tris-egg yolk and 47% in Bioexcell (soya milk-based) extenders (Akhter et al., 2010), 41.5% in egg yolk-based and 56% in soya bean-based extenders (Akhter et al., 2011a), 41.5% in egg yolk-based and 53.5% in LDLs-based extenders (Akhter et al., 2011b).
The success rate of pregnancy in mares that were inseminated either hysteroscopically or conventionally using frozen-thawed epididymal sperm was 18% versus 8% (Morris et al., 2002). Pregnancy rate in mares could only be increased by deposited the epididymal sperm surgically in the region of utero-tubal junction in order to achieve equal success rate of pregnancy using ejaculate sperm (Morris et al., 2000). Eventhough, recent report showed a similar pregnancy rate using epididymal sperm (55.8%) versus ejaculate sperm (53.8%) in sheep (Álvarez et al., 2012).
Some problems in buffalo AI program are from female and male sides. Silent heat phenomenon that is common in female is caused by low hormonal level. This condition was generated the difficulties in estrous detection, un-appropriate time for insemination, and the failure of fertilization. Small uterine body size prevent the sperm to be introduced into one uterine horn could also be a reason for low conception rates to AI (Sansone et al., 2000). Due to that we understand that high quality of sperm is absolutely needed to increase the success rate of AI. Although, we optimist that frozen-thawed epididymal sperm is suitable to be used in spotted buffalo AI program.

CONCLUSION
Both tris-based and citrate-based extenders have equal ability in maintaining the post-thawed spotted buffalo epididymal sperm quality and fertilizing capacity in artificial insemination program. It is suggested that these two chemically defined extenders could be used to increase the efficiency and reduce the cost in sperm processing and cryopreservation in spotted buffalo conservation program.

ACKNOWLEDGEMENT
This study was funded by Indonesian Ministry of Education and Culture, Directorate General of Higher Education for Foreign Research Networking Project No. 447/SP2H/PP/DP2M/VI/2010. The authors wish to acknowledge the head and staff of Animal Division of North Toraja Government for supporting this study and collecting samples. Finally, we wish to thank all of the farmers and owners that have provided the buffalo samples for this study. Without their help, this study wouldn't be possible.