Successful Intracervical Insemination and Characteristics of Anoa (Bubalus SP.) Parturation Behavior in Captivity

Anoa, which is the endemic animal in Indonesia, its population is unevitably decreasing, therefore anoa included in the list of endangered species by International Union for Conservation of Nature (IUCN). The experiment was aimed to apply the artificial insemination (AI) technique for anoa and to examine their parturition behavior. The experiment involved 2 males and 5 females anoa at Taman Safari Indonesia Bogor. Semen was collected by electroejaculator, then evaluated and diluted in Tris-egg yolk extender. The anesthetized estrous females were inseminated intracervically with extended semen (100x106 sperm/1.0 mL). Females which did not show estrous signs during 2-3 cycles after AI were predicted pregnant, and therefore confirmed by ultrasonography. As a comparison, another predicted-pregnant female after natural mating was scanned. The results showed that intracervical AI resulted pregnancy in one female, and the gestation period of anoa was ranged from 313 d (AI) to 324 d (natural mating). There were three stages of parturition process was observed: Stage 1 was characterized by the abdominal contraction and amniotic membrane rupture, Stage 2 was characterized by the fetal expulsions, and Stage 3 was characterized by the placental expulsions and ingestion of placenta by the dam. The duration of each stage of parturition was 6-8 h (stage 1), 30-60 min (stage 2), and 15-180 min (stage 3). It is concluded that AI technique was applicable to anoa in captivity, and the parturation behavior of anoa was comparable to buffalo and cattle. However, the fetal delivery occurred when the dam was in standing position and the dam ingested the placenta.


INTRODUCTION
Anoa is an endemic animal in Indonesia found in Sulawesi and its offshore islands. There are two species of anoa, the lowland anoa (Bubalus depressicornis Smith, 1827) and the mountain anoa (Bubalus quarlesi Ouwens, 1910) (Burton et al., 2005a). The population of anoa in the wild is in rapidly decline, so both of them are classified as endangered by IUCN and included into Appendix I of the CITES (Burton et al., 2005a).
Both species of anoa are vulnerable due to their restricted range of habitat and extinction possibilities from several threats by illegal hunting, habitat loss and fragmentation, and possible natural catastrophes (Burton et al., 2005b). These threats cause the anoa become isolated in small populations in the fragmented habitat or lived ons in the fragmented habitat or lived ns in the fragmented habitat or lived in the conservation area that may cause to the loss of genetic diversity and increasing extinction risks (Burton et al., 2005a). Therefore, the management of the anoa population in captivity is important. Unfortunately, until present, anoa breeding efforts in captivity have not anoa breeding efforts in captivity have not breeding efforts in captivity have not been succeed, that might be caused by their aggressivity, gressivity, ressivity, solitary, wildness, and monogamy, as well as the limita-ary, wildness, and monogamy, as well as the limita-, wildness, and monogamy, as well as the limitation of their reproductive biology data.
Several studies have been conducted in many aspects of the anoa, but the study on their reproduction is still limit; they include the sexual behavior (Feer, 199�), , 1994), 1994), taxonomy (Burton et al., 2005a), potency of anoa as a farm animals (Kasim, 2002), semen collection and its characteristics (�udi �udi udi et al., 2009), and sperm cryopreservation (�udi �udi udi et al., 2011). However, it has not been reported the application of assisted reproductive techniques (ART) especially on the artificial insemination (AI) and also the behavior during parturation, as essential stages for the success of breeding in anoa in captivity. In fact, this information might be very important to improve their management and handling (the dam and the fetus) during peri-partum period.
The characteristics of anoa behavior are soliter, aggressive, and tend to be monogamus. These characteristics may result in many problems in the future caused by the limitation of population. Therefore, application of ARTs (i.e. AI technique) in anoa is very important. In addition, sperm cryopreservation as a stage of AI program, will be a genetic resource of scarce animal, and furthermore can be used in other ARTs application, i.e. . in vitro fertilization and cloning (Holt & Pickard, 1999;Morrow et al., 2009). The successful of AI application in many wild animals have been reported, for examples in puma and deer (Holt & Pickard, 1999), African white rhino (Hermes et al., 2009), and llama and zebra (Adams et al., 2009). Application of AI technique in wild animals in captive breeding may prevents inbreeding incidence caused by the lacking of the population or genetic diversity; inbreeding may cause increase the risk of testis development defects with consequent infertility, embryonic loss, and decreases the adaptation capacity (Hold & Pickard, 1999;Commizoli et al., 2000).
The birth of a healthy calf is basic requirement for the successful of the captive breeding. Therefore, detection of some complications during parturation and helping them as soon as possible are important. Calves from difficult parturations show a clearly increased mortality during the first 2� h after parturation (Wehrend et al., 2006). Understanding the characteristics behavior of parturation is necessary to improve parturation management. In wild animals, there are technical limitations linked to various anatomy, physiology, and behavior (Commizoli et al., 2000). This experiment was conducted to apply the AI technique for anoa breeding and to apply the AI technique for anoa breeding and to ply the AI technique for anoa breeding and to AI technique for anoa breeding and to AI technique for anoa breeding and to study the characteristics of parturation behavior in anoa in captivity.

Experimental Animals
The experiment was conducted in Taman Safari Indonesia Bogor West Java, using 7 anoas: 2 males and 5 females (i.e. Bu, Ay, Ta, Lu, and Ke). All anoas were s were were clinically in a good condition and were maintained with standard captive practices.

Semen Collection, Evaluation, and Processing
Semen was collected from anesthetized animals etized animals ized animals by using electroejaculator (modified from Paris et al., 2005) connected to an electrostimulator AC 100 Hz and 1.50 cm diameter of rectal probe with four circular electrodes (Fujihira FHK-Japan). The probe was lubricated with jelly (KY Lubricating Jelly ® , PT �ohnson & Johnson-Indonesia) and inserted into the rectum with the electrode ventrally oriented in close apposition to the assessory glands. The electroejaculation sequence sory glands. The electroejaculation sequence ory glands. The electroejaculation sequence consisted of six series was separated by 5-s rest periods. Each stimulus was consisted of three 5-s stimuli at each of voltages (3, 5, 7, 9, 11, and 12 V).

Intraservical Insemination
The synchronized females (n= �) showing estrous signs were inseminated intracervically (Verberckmoes et al., 2001;Kershaw et al., 2005) using extended semen with insemination dose of 100 x 10 6 sperm/ml. Insemination was conducted in approximately 50-60 h after luprostiol injection under anesthetized condition etized condition ized condition using AI pipette (Minitub �mbH-�ermany) commonly used for both sheep and goats. A vaginal speculum was inserted into the vagina to locate os cervix by means of a light source fixed on the speculum, and semen was deposited in the maximum depth of cervical penetration by the pipette (Batista et al., 2009). During insemination, the perineal region of female was lifted for around 5 min after semen deposition to help the sperm to enter the uterus.

Pregnancy Diagnosis
One female (Lup) that did not show estrous signs during 2-3 of estrous cycles after insemination was predicted pregnant, and therefore was confirmed by ultrasonography (US�). A transrectal scanning using a 7.5 MHz linear probe (Aloka SSD-500, Aloka Co. Ltd.-Japan) was performed for pregnancy diagnosis under anesthetized condition. Examination was done for etized condition. Examination was done for ized condition. Examination was done for around 6 mo after insemination. The active surface of the transducer (where piezoelectric crystals are located) was lubricated with jelly and inserted through anal opening, before progressing cranially along the rectal floor to overlie the reproductive tract (Vyas et al., 2002). The lubricated face of the transducer was pressed firmly towards the ventral rectal wall to ensure the transmission of ultrasound waves through rectal mucosa into pelvic and abdominal viscera. As a comparison, another female (K) that predicted pregnant after 7 mo of natural mating was scanned.

Observation of Parturation Behavior
Parturation behavior was observed from a distance of 2-5 m from outside the yard or pen, starting from 6 mo gestation (modified from Wehrend et al., 2006). Observation was done in 2 females positively diagnosed pregnant by US�. The following parameters were documented: feeding behavior, the udder and teats swelling, vulva swelling, excretion of cervical mucus, restlessness, lying down and getting up, vocalization, abdominal contraction, kyphosis position, and also expulsion of amniotic sac, fetus, and placenta. Observation was carried out during the day time, from 07.00 to 17.00 o'clock. When the behavior changed that indicate the partum changed that indicate the partum indicate the partum would be occurred soon, observation was done 24 h per d. During parturation process, observation focused on the behavior in each stage of parturation. The behavior was recorded and classified into 3 stages: the first stage (preparation/cervical dilatation), the second stage (fetal delivery), and the third stage (placental delivery) (Senger, 2005;Wehrend et al., 2006).

Successful of AI Application
Present study showed that AI application in anoa resulted in one out of four female was pregnant and produce a live male calf. However, the efficiency was still low (25% of pregnancy rate, PR and 8.0 of service per conception, S/C) ( Table 2). Confirmation of the pregnant female using US� at around 6 mo of gestation showed the eye balls, sculls, ribs, and other organs of the fetuses, as well as the placentomes (Figure 1).
The results of this study suggested that AI application in anoa was still inefficient compared to the farm animals. In cattle, AI application is efficient if the PR application is efficient if the PR is efficient if the PR is higher than 75% and S/C is 1.2-2.0 (Toelihere, 1993). Verberckmoes et al. (2001) reported that intracervical insemination in sheep resulted in 56%-75% PR, but Batista et al. (2009) showed only �2.8% PR. In In fallow deer, intracervical insemination resulted PR of 62.9% PR (using frozen semen) to 76.3% (using fresh semen) (Jabbour et al., 1993). However, AI application in many others wild mammals resulted low pregnancy rate. For examples, transcervical AI in African white rhino (Ceratotherium simum) resulted in a pregnancy of two females and produced a healthy offspring (Hermes et al., 2009), and in South American camelids resulted in 39% (frozen semen) to 51% PR (fresh semen) (Adams et al., 2009).
Successful AI application in wild mammals established by understanding of species-characteristics of the anatomy and physiology of reproduction, stress management, sperm quality and quantity, oocytes quality, dates of insemination, semen deposition, gametes trans-portation system, and early embryonic loss (Toelihere, 1993;Durrant, 2009;Hermes et al., 2009;Morrow et al., 2009). The lower pregnancy rate found in this experiment might have been due to insufficient sperm quality in terms of low progressive motility (52.5%) and high level of total sperm abnormality (31.8%) that was reported previously (�udi et al., 2009). The quantity of total sperm number (100 x 10 6 sperm/ml) may below the minimum threshold required to achieve fertilization in this species. Hermes et al. (2009) reported that AI in African white rhino with 135 x 10 6 motile sperm was failed, but when the dose was increased up to 500 x 10 6 motile sperm resulted a pregnancy. In sheep, the maximum depth of cervical penetration during intracervical insemination was affected by both cervical grade of canalis cervicalis and the dates of estrous phase, and those factors may affect the result of AI application (Kershaw et al., 2005). Unfortunately, the optimum mating time and the anatomy of the cervix uteri of anoa have not yet been understood. Therefore, to improve the pregnancy rate in anoa in captivity, more information about the reproductive biology, animal handling, determine of the optimum mating time, sperm preservation and cryopreservation, and also insemination dose and semen deposition are required.

The Length of Gestation Period
Present result showed that the gestation length of anoa was 313 d (resulted of AI) and 324 d (resulted of natural mating) ( Table 2). The result is longer than that   Kasim (2002) previously, 275-315 d. The gestation period of anoa was comparable to swamp buffalo (310 d) and mare (330 d), and therefore, it was longer than in cattle (278 d) and in goat (150 d) (Jainudeen & Hafez, 2000). The length of gestation period of the primipara (K) with a female fetus (32� d) was longer than the multipara (L) with a male fetus (313 d).
Pregnancy period is variable in breeds, species, and individual within species, because of genetic factors, parity, age, nutrition, litter size, and sex of the fetus (Toelihere, 1993;Hutchin et al., 1996;�ainudeen & Hafez, 2000). Hutchin et al. (1996) reported that male fetuses may extend the gestation length (for example in cattle and sheep), and the increased the fetal number will shorten it (for example in cattle, canidae, and mice). The date of conception during breeding season also affect the length of gestation period of red deer (Cervus elaphus), may be related with the feed intake and quality (Scott et al., 2008).

Characteristics of Parturation Process
At around 7 mo of gestation, occasionally in the morning, the brownish mucus was appeared hanged out from the vulva, and the color changed to reddish-dark brown at around 9 mo of gestation. At that time also appeared swelling of the teats and labia of the vulva. A week toward parturation, the mucus was expelled more frequently and voluminous, the dams was occasionally laying down inside and the feet were stretched, and aparted from her group. During 1-2 d toward parturation, the dams showed restlessness (getting up and lay- Figure 2. The serial photos described the parturation process in anoa and its behavior observed in captivity; showed the cervical mucus hanged out from vulva (A), abdominal contraction and kyphosis (B), expulsion of the amnotic membrane (C), expulsion of both fetal foreleg (D), the dam was licking the fetus (E) and eating the placenta (F).

Stages of parturation Behavioral elements Duration
Stage 1 (preparation) -cervical mucus was excreted, hang out from the vulva -abdominal contraction, more frequently and powerful when the amniotic sac was expelled -occasionally kyphosis position and vocalization -expulsion of fetal foreleg (in the amniotic sac), -the dams showed restlessness (getting up and lying down, more movement) -amniotic membrane was ruptured -abdominal contraction: 6 to 8 h -amniotic membrane expulsion: 2 h Stage 2 (fetal delivery) -the calves was visible in the rima vulvae -the fetal position was antero-dorsal (fetal vertebrae in upper site) to delivery canal -fetal delivery, the dam was in standing position 30 min (L) to 1 h (K)

Stage 3 (placental delivery)
-the dams licked the fetuses -expulsion of placenta, by the maternal contraction (K) or pulled out by the dam (L) -the dams ate the placenta immediately after delivered 15 min (L) to 3 h (K) ing down, more movement), decreasing of appetite, and more vocalization. These behavior are common in the last second semester of pregnancy (Jainudeen & Hafez, 2000), particularly in pasturing ungulates (Wodzicka-Tomaszewska et al., 1991).
Entering to the first stage of parturation (preparation stage), the anoa showed abdominal contraction, moving around the pens, and expulsion and rupture of amniotic membrane (Table 3, Figure 2). The second �igure 2). The second . The second stage of parturation (fetal delivery) was characterized by the appearance of the calf in the rima vulvae, and fetal expulsion. The last stage of parturation (placental delivery) was characterized by expulsion and delivery of placenta, and the dams lick the fetus and eat the placenta. Duration of each stage of parturation was 6-8 h in the first stage, 30-60 min in the second stage, and 15-180 min in the third stage. Fetal expulsion occurred while the dams were in standing position. One female (L) pulled out the placenta which was hanging out from the vulva with her mouth and then ate it, whereas another female (K) ate the placenta that was already fell to the floor. This maybe an instinct behavior in wild to escape from natural catastrophes, which was still exhibit in anoa lived in captivity.
The duration of parturation varied among species, and affected by many factors such as fetal weigh, fetal position, litter size, the sex of fetus, parity, breeds and species, and pelvic size of the dams (Toelihere, 1993;Jainudeen & Hafez, 2000). In cattle and buffalo, the duration of the first, second, and third stages of parturation were 2-6 h, 0.5-1 h and 8-12 h, respectively; whereas, in sheep were 2-6 h, 0.5-2 h and 0.5-8.0 h, respectively and 0.5-8.0 h, respectively 0.5-8.0 h, respectively (�ainudeen & Hafez, 2000). The present result suggested that duration of parturation in the anoa was comparable to buffalo and cattle.

CONCLUSION
AI technique was applicable for anoa breeding in captivity. The gestation period of anoa was about 313 and 324 d. Gestation period and characteristics of parturation behavior of anoa were comparable to buffalo and cattle. The fetal delivery occurred when the dam was in standing position and the dam ingested the placenta.