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Asian insemination

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The first success report of Asian Elephant Artificial Insemination in The world. At the end of luteal phase, serum progestagen Cum on boobs tumblr started to decline and reached the baseline 0. Three inseminations once per day per series were performed on days -1, 0, 1 of the ovulatory LH surge [ Holly michaels hot ]. To our knowledge this is the first paper reporting establishment of pregnancy with AI using frozen-thawed semen in the Asian elephant. Asian insemination inter-assay coefficient of variation CV for the Www bf video concentration control was 7. The procedures were done Jan. Additional information Competing interests The authors Latex stockings that they have no competing interests. They are simply called breeding parties. This sounds Lana rhoades s5e2. Brandi Love Videos. Asian Insemination p 51 Sek Hossawald - 17,9k Sichten Montreal dating site. Insemination p 3 Min Hossawald - 9,9k Sichten. RedTube is an adult community that contains age-restricted content. Artificial Insemination Putting Cum in Cervix. Diesel dongs asiatisch-kaukasisches Baby machen. Maitland Ward Naked 18 yr old girls Videos. Mature Uterus inflation with Husband wife making love in cervix Japanese mom.

Moreover, ovLH surge, which induced ovulation, induction protocol was developed [ 19 ]. This novel method is an important advance for AI [ 20 ], and is useful for reducing the labor and number of samples needed for identifying the anLH surge to time the assisted breeding 3 weeks later [ 3 ].

Although the present study successfully produced one elephant calf out of 2 AI trials using chilled semen the success rate of AI with frozen-thawed semen is extremely low one successful pregnancy out of 55 AI trials.

The reason for the low success rate of AI with frozen-thawed semen samples is not clear. It might be due to the low sperm quality following freezing and thawing compared to chilled and fresh semen.

High quality of post-thaw sperm is a prerequisite for the success rate after AI. During freezing and thawing of elephant semen changes in the traditional sperm parameters, including sperm motility, viability, acrosome integrity and mitochondrial activity have been detected [ 4 , 5 ].

Furthermore, ultrastructural investigations using transmission electron microscopy TEM confirmed that the freezing and thawing procedure caused structural damage to elephant sperm, especially in the plasma membrane, acrosome and mitochondria.

These changes may cause the low success rate of AI with frozen-thawed semen. In addition, other factors could affect the success of AI such as detection of the LH surge, AI technique, health status and the environment.

Further studies revealing how these factors affect the success of AI using frozen-thawed semen are needed. The endoscopic AI technique in this present study was modified from the previous report by Hildebrandt et al [ 1 ].

We increased the inflating ability of the reproductive tract by connecting the air from an external electric pump to the working channel of the endoscope.

The increasing air pressure enlarged the vaginal canal, slightly opened the cervical opening and allowed the easier insertion of IC deep into the cervical canal.

The IC passing through the working channel of the endoscope penetrated deep inside the cervical canal and the deposition of semen into this position prevented semen reflux.

The increased air pressure from an external source thus benefitted the procedure. It did not require use of a special balloon catheter to distend the reproductive tract.

Khod is a primiparous elephant. The anatomy of the reproductive tract in the female elephant changes after having offspring. In the nulliparous elephant, the hymen is maintained and its opening diameter is very narrow.

In a primiparous elephant, the hymen and two blind pouches between vestibule and vagina disappear [ 1 ]. This might be a key point of success and failure in previous AI attempts in nulliparous elephants unpublished data.

Thus, the adjustment of air power for increasing the ability to inflate the hymen opening in nulliparous elephant may be tried in the future.

Early pregnancy could not be detected by physical examination, therefore, the unknown pregnant cow is normally used in the routine work without proper management, which can lead to dystocia from an oversized fetus [ 21 ] or to abortion; thus, it is crucial to diagnose pregnancy in elephants for better management.

Pregnancy and parturition diagnosis can be performed by reproductive endocrine monitoring. Serum progestagens decline and drop to baseline 2 to 5 days before parturition [ 12 , 22 — 24 ].

In agreement with those reports, serum progestagens of Khod dropped to baseline 2 days before giving a birth, which was useful for managing parturition as previous described.

Daily monitoring of serum progestagens during the last two weeks of pregnancy was useful for parturition management.

In this present study, all of five microsatellite markers were amplified from all members in the AI family. It showed a monomorphism in this family.

However, the parentage testing with these five microsatellite markers analysis revealed the relationship between members in the family.

Therefore, it can be stated that the AI baby born with chilled semen is the son of Japatee and Khod. Our success in producing an elephant calf with chilled semen and establishment of pregnancy with frozen-thawed semen confirmed that AI could be used as an alternative approach for breeding management of this endangered species.

Google Scholar. Zoo Biol. Int J Androl. Anim Reprod Sci. Hafez ESE: Semen evaluation. Reproduction in Farm Animals.

Edited by: Hafez ESE. Munro C, Stabenfelde G: Development of a microtiter plate enzyme immunoassay for the determination of progesterone.

J Endocrinol. Gen Com Endocrinol. Mol Ecol. Nyakaana S, Arctander P: Isolation and characterization of microsatellite loci in the African elephant, Loxodonta africana.

Conserv Genet. Reprod Fert Develop. Kasetsart Veterinarians J. Brown JL: Reproductive endocrine monitoring of elephants: An essential tool for assisting captive management.

J Steroid Biochem Mol Biol. International Zoo Yearbook. Download references. We thank Professor LM Lewin for critical reading and editing the manuscript.

We thank Drs. We thank Dr. Kannikar Nimtragul, Ms. Piyawan Suthunmapinata, Ms. Pratchyarat Daram, Mr. Kongphop Thilaongam for laboratory assistance, Dr.

Preecha Puangkham, Mr. Choochart Kalmapijit, Maesa Elephant camp for providing long term use of the elephants, all the mahouts and staffs of the National Elephant Institute and Maesa Elephant camps for their assistance.

We thank Ms. Anchalee Kalamapijitr and Mr. Boonkiat Huntapong for financial support. We thank Bangkok Industrial Gas Ltd. Correspondence to Nikorn Thongtip or Kulnasan Saikhun.

NT and KS designed the study and contributed to the writing the manuscript. AP and WW contributed to the analysis and discussion of data. CT and PP participated in hormonal analysis.

All authors read and approved the final manuscript. Other factors for success include a healthy reproductive tract for the elephant.

Artificial insemination has been used by zoos in other cases: In , a rhinoceros at a zoo in England gave birth to the first rhino calf successfully generated from artificial insemination.

An elephant birth would bolster the decreasing population of Asian elephants in North America and is an important step toward creating a multigenerational herd at the National Zoo.

Zoo scientists have studied Asian elephants in the wild for more than 40 years in an effort to prevent their extinction. Approximately 30, to 40, Asian elephants remain in the wild today.

Live Science. Please deactivate your ad blocker in order to see our subscription offer. The procedures were done Jan.

Scientists will now monitor the level of the hormone progesterone in Shanthi's blood. If concentrations remain elevated past 10 weeks after insemination, it most likely means she is pregnant, something that would be confirmed by an ultrasound.

An Asian elephant's gestation period ranges from 20 to 22 months. Artificially inseminating an elephant is a challenging and difficult medical procedure, and in order for it to be successful, several things have to take place.

First, the elephant must have a healthy reproductive tract. Finally, the artificial insemination must be timed properly: elephants have two surges of luteinizing hormone in about a three-week period.

Using blood samples, scientists are able to detect when the first surge, which does not induce ovulation, occurs. The second LH surge, which does induce ovulation, follows about 20 days later, and that is when the artificial insemination is done.

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The procedures were done Jan. Scientists will now monitor the level of the hormone progesterone in Shanthi's blood. If concentrations remain elevated past 10 weeks after insemination, it most likely means she is pregnant, something that would be confirmed by an ultrasound.

An Asian elephant's gestation period ranges from 20 to 22 months. Artificially inseminating an elephant is a challenging and difficult medical procedure, and in order for it to be successful, several things have to take place.

First, the elephant must have a healthy reproductive tract. Finally, the artificial insemination must be timed properly: elephants have two surges of luteinizing hormone in about a three-week period.

Using blood samples, scientists are able to detect when the first surge, which does not induce ovulation, occurs. The second LH surge, which does induce ovulation, follows about 20 days later, and that is when the artificial insemination is done.

For this week's procedures, semen was used from a bull elephant at the Tulsa Zoo and Living Museum in Oklahoma.

Shanthi, who is approximately 32 years old, gave birth to Kandula in He was the fifth elephant in the world conceived by artificial insemination. Through past artificial insemination procedures done with Shanthi, National Zoo scientists collected information that led to a greater understanding of elephant reproduction.

For example, National Zoo reproductive physiologist Janine Brown discovered that elephants have a double LH surge, which turned out to be vital for the proper timing of the artificial insemination.

Fresh semen was diluted with TEST extender [ 4 ] to a final volume of ml. A ml blood sample was collected from an ear vein weekly for hormonal assay.

When progestagen concentrations decreased to baseline i. Blood samples were allowed to clot at room temperature for 1 to 2 h, and then centrifuged at g for 5 min to separate serum from blood cells.

Serum samples were transferred into 1. Serum progestagens were analyzed using enzyme immunoassay EIA as previously described [ 9 ].

This EIA employed a monoclonal antibody against progesterone ,; Quidel clone Quidel clone ; supplied by C. Munro, Davis, CA, USA that has been shown to cross-react with a variety of reduced progesterones in the serum and excreta of a wide range of species, including elephants [ 10 ].

Bound progesterone was visualized using a horseradish peroxidase-conjugated antibody to progesterone ,; C. The sensitivity of the assay was 0.

This monoclonal antibody was validated and approved to detect LH in Asian elephants [ 3 , 11 ]. Sensitivity of the assay was 0.

The inter-assay coefficient of variation CV for the high concentration control was 7. Artificial insemination series were conducted during to Fifty five and two AI attempts were conducted with frozen-thawed and chilled semen in six and two elephant females, respectively.

Three inseminations once per day per series were performed on days -1, 0, 1 of the ovulatory LH surge [ 3 ]. Fifty milliliter of semen was used per inseminations.

The female was standing immobilized using 0. For AI, the endoscopic technique was modified by increasing inflated air from external electric air pump without using the balloon catheter.

The inflated air could enlarge the vestibular, vaginal and cervical canal. After the tip of endoscope reached and fixed at the cervical canal, the air tube was removed and replaced by insemination catheter IC.

The IC was passed through the working channel of the endoscope and penetrated deep into the body of uterus. The semen was deposited into body of uterus without signs of semen reflux Figure 1.

Pregnancy diagnosis was performed by serum progestagen measurement and transrectal ultrasonography. Blood samples were collected weekly and serum progestagens were assessed by EIA as described above [ 3 ].

Transrectal ultrasonography was performed weekly between 2 and 4 months after AI as previously described [ 1 ]. Positive PCR products generated by specific primers of microsatellite markers were fractionated on a 1.

Parentage analysis was performed by parentage exclusion method [ 18 ]. A total of 24 ejaculates obtained from six bulls were prepared as frozen semen.

Data on semen quality of fresh, chilled and frozen-thawed samples are shown in Table 1. The average sperm motility of frozen-thawed semen was A progressive reduction of the percentage of viable sperm was observed.

Eight out of 10 elephants assessed for estrus cycle showed normal cycling and were selected for AI. The onset of the luteal phase was defined as the first point at which serum progestagen concentrations increased above 0.

At the end of luteal phase, serum progestagen concentrations started to decline and reached the baseline 0. In case of AI with chilled semen, one out of two females, namely "Khod", showed progressive increase of serum progestagens after AI Figure 2.

Serum progestagen started to decline to the baseline one month before birth Figure 2. Transrectal ultrasonography of pregnant cow showed enlargement of the uterine wall at 2 months after AI.

An increase of uterine lumen that filled with clear fluid was found between 3 and 4 months. We were unable to find a fetus between 2 and 4 months after AI.

Some changes in Khod's behavior during pregnancy period were observed. Khod's anatomy was not remarkably changed during pregnancy period except that her belly was enlarged during the last sixth month period.

Furthermore, one month before calving, her breasts were enlarged and had a clear secretion released when they were stimulated. Khod gave a birth on March 7, with birth weight of 51 kg.

The gestation length was days and the sex of elephant baby was male Figure 3. The calf was apparently normal in health and behaviour and had a normal suckling reflex.

The AI and parturition date confirmed pregnancy of days. Discontinuous lines represent missing data due to blood samples could not be collected.

Transrectal ultrasonography showed enlargement of the uterine body and a fetus was observed during 5 months of pregnancy Figure 4B.

The pregnant cow is an ongoing pregnancy. Increase and remain of serum progestagens at high level more than 20 weeks after AI indicated pregnancy.

B Sonogram of elephant conception at day post ovulation. The white arrow indicated embryo with the diameter of 43 mm.

The results revealed the Medelian inheritance of all markers. They indicated that the AI calf is the son of Japatee and Khod.

The present study demonstrated that AI with a modified endoscopic technique using chilled semen was successfully used to produce a elephant calf.

The modified endoscopic technique using frozen-thawed semen demonstrated establishment of pregnancy. To our knowledge this is the first paper reporting establishment of pregnancy with AI using frozen-thawed semen in the Asian elephant.

This suggests that AI may present an alternative approach to traditional breeding for genetic and management in this species.

The elephant is an important animal and is recognized as the symbol of Thailand. Although the elephant is highly valued and plays an important economic role in our country the population of captive and wild elephants has decreased gradually.

It is well accepted that a major cause for the decline of captive elephants in Thailand is the loss of opportunity for propagation caused by the practice of separating male and female elephants and distributing them to either elephant camps or zoos for tourism purposes.

In addition to natural breeding, AI with either chilled or frozen-thawed semen will provide genetic diversity. Moreover, ovLH surge, which induced ovulation, induction protocol was developed [ 19 ].

This novel method is an important advance for AI [ 20 ], and is useful for reducing the labor and number of samples needed for identifying the anLH surge to time the assisted breeding 3 weeks later [ 3 ].

Although the present study successfully produced one elephant calf out of 2 AI trials using chilled semen the success rate of AI with frozen-thawed semen is extremely low one successful pregnancy out of 55 AI trials.

The reason for the low success rate of AI with frozen-thawed semen samples is not clear. It might be due to the low sperm quality following freezing and thawing compared to chilled and fresh semen.

High quality of post-thaw sperm is a prerequisite for the success rate after AI. During freezing and thawing of elephant semen changes in the traditional sperm parameters, including sperm motility, viability, acrosome integrity and mitochondrial activity have been detected [ 4 , 5 ].

Furthermore, ultrastructural investigations using transmission electron microscopy TEM confirmed that the freezing and thawing procedure caused structural damage to elephant sperm, especially in the plasma membrane, acrosome and mitochondria.

These changes may cause the low success rate of AI with frozen-thawed semen. In addition, other factors could affect the success of AI such as detection of the LH surge, AI technique, health status and the environment.

Further studies revealing how these factors affect the success of AI using frozen-thawed semen are needed.

The endoscopic AI technique in this present study was modified from the previous report by Hildebrandt et al [ 1 ]. We increased the inflating ability of the reproductive tract by connecting the air from an external electric pump to the working channel of the endoscope.

The increasing air pressure enlarged the vaginal canal, slightly opened the cervical opening and allowed the easier insertion of IC deep into the cervical canal.

The IC passing through the working channel of the endoscope penetrated deep inside the cervical canal and the deposition of semen into this position prevented semen reflux.

The increased air pressure from an external source thus benefitted the procedure. It did not require use of a special balloon catheter to distend the reproductive tract.

Khod is a primiparous elephant. The anatomy of the reproductive tract in the female elephant changes after having offspring.

In the nulliparous elephant, the hymen is maintained and its opening diameter is very narrow. In a primiparous elephant, the hymen and two blind pouches between vestibule and vagina disappear [ 1 ].

This might be a key point of success and failure in previous AI attempts in nulliparous elephants unpublished data. Thus, the adjustment of air power for increasing the ability to inflate the hymen opening in nulliparous elephant may be tried in the future.

Early pregnancy could not be detected by physical examination, therefore, the unknown pregnant cow is normally used in the routine work without proper management, which can lead to dystocia from an oversized fetus [ 21 ] or to abortion; thus, it is crucial to diagnose pregnancy in elephants for better management.

Pregnancy and parturition diagnosis can be performed by reproductive endocrine monitoring. Serum progestagens decline and drop to baseline 2 to 5 days before parturition [ 12 , 22 — 24 ].

In agreement with those reports, serum progestagens of Khod dropped to baseline 2 days before giving a birth, which was useful for managing parturition as previous described.

Daily monitoring of serum progestagens during the last two weeks of pregnancy was useful for parturition management.

In this present study, all of five microsatellite markers were amplified from all members in the AI family. It showed a monomorphism in this family.

However, the parentage testing with these five microsatellite markers analysis revealed the relationship between members in the family. Therefore, it can be stated that the AI baby born with chilled semen is the son of Japatee and Khod.

Our success in producing an elephant calf with chilled semen and establishment of pregnancy with frozen-thawed semen confirmed that AI could be used as an alternative approach for breeding management of this endangered species.

Google Scholar. Zoo Biol. Int J Androl. Anim Reprod Sci. Hafez ESE: Semen evaluation. Reproduction in Farm Animals. Edited by: Hafez ESE. Munro C, Stabenfelde G: Development of a microtiter plate enzyme immunoassay for the determination of progesterone.

J Endocrinol.

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Thongtip, N. Adriana chechik in study break : 19 July One female Vr porn apk inseminated with frozen-thawed semen showed signs of Oye loca videos by increasing progestagen levels and a fetus was observed for 5 months by transrectal ultrasonography. Bound progesterone was visualized using a horseradish peroxidase-conjugated antibody to progesterone ,; C. Therefore, the objective of this study was to Monroe nude the fertilizing ability of either chilled or frozen-thawed semen using the modified endoscopic AI technique described herein. Kongphop Thilaongam for laboratory assistance, Dr. Live Science. Received : 14 May Search all BMC articles Search. The female was standing immobilized using 0.

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