Proceedings of The 7 Wildlife Assisted Reproductive ... · “The 7th Wildlife Assisted Reproductive Technology (ART) Workshop” This workshop is aimed to establish one of the stages

the 7th Wildlife Assisted Reproductive Technology (ART) Workshop 2016

Proceedings of

The 7th Wildlife Assisted Reproductive Technology (ART) Workshop

“Reproductive Biotechnology: from Basic to Applications”

March 7-11, 2016


Proceedings of

The 7th Wildlife Assisted Reproductive Technology (ART)

Workshop

March 7-11, 2016


Welcome Message

By Associate Professor Dr. Chuchat Kamollerd

Dean of Faculty of Veterinary Medicine,

Khon Kaen University

--------------------------------------------------------------------------------------------------------------------------------

Distinguished participants

Ladies and Gentlemen.

On behalf of the Faculty of Veterinary Medicine, Khon Kaen University, it is my great

pleasure to you all a very warm welcome to the 7th Wildlife Assisted Reproductive Technology

(ART) Workshop 2016, which is the joint conference of the Zoological Park Organization and

Khon Kaen University. The conference is arranged during 7th-11st March 2016, the scientific

session held at the Faculty of Veterinary Medicine, Khon Kaen University and the workshop held

at Khon Kaen Zoo, Khon Kaen, Thailand.

It is a very great opportunity to have the collaboration with the Zoological Park

Organization and Khon Kaen Zoo. The conference will dedicate to provide scientific forum focus

on wildlife reproduction and ART for scientists, veterinarians, graduate and students to learn and

exchange more knowledge. I hope that we all will enjoy the conference and best wish for the

successful workshop.

I wish that you will have a pleasant time in Khon Kaen. I hope that the joint symposium of

Zoological Park Organization and Khon Kaen University will continue for the future and have

a strengthen relationship in animal reproductive science and technologies.

Thank you very much.


Opening Remarks

By Mr.Benjapon Nakprasert

Director of Zoological Park Organization

under the Royal Patronage of H.M. the King

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Distinguished participants

Ladies and Gentlemen

On behalf of the organizing committee of the 7th Wildlife Assisted Reproductive

Technology (ART) Workshop 2016, I am very pleased to welcome colleagues, friends,

veterinarians, scientists, students and exhibitors who participating in the joint conference of

Khon Kaen University and Zoological Park Organization. This conference is a great occasion for

researchers and veterinarians whose works focus for animal reproductive biology to share

knowledge and experiences, which in turn will be a great contribution to the wildlife

conservation.

The conference theme “Reproductive Biotechnology: from Basic to Applications”

will offer relevant delegates to have opportunities to review fundamental reproductive science,

access novel research finding and technologies for wildlife. The conference features special

lectures given by experts in the field and sessions for free communication. The conference also

provides a hand on workshop for researchers in the field to practice and share the experience for

building up network in future.

I wish you have productive scientific forum, successful workshop and enjoy the time

during the conference.


Organizing committee

1. Benjapon Nakprasert Advisor

2. Assoc Prof.Dr. Chuchart Kamollerd Advisor

3. Sumate Kamolnorranath Advisor

4. Tanachon Kensingh Advisor

5. Dr. Ampika Thongphakdee Chairperson

6. Asst Prof. Sarawut Sringam Vice Chairperson

7. Asst Prof. Dr. Vibuntita Chankitisakul Chairperson of Scientific Committee

8. Chavin Chaisongkarm Venue Committee

9. Nudthakamol Kajornklin Secretary

10. Saifon Yapila Assistant secretary

11. Supalak Kaitsomboon Registration Committee

12. Sunthita Karoon Treasurer

13. Dr.Saksiri Sirisathien Moderator

14. Asst Prof.Dr.Wuttigrai Boonkum Moderator


“The 7th Wildlife Assisted Reproductive Technology

(ART) Workshop”

This workshop is aimed to establish one of the stages where reproductive scientist, veterinarian and relevance persons can join together sharing, talking, and giving their experiences about reproductive science and technology to each others.

The dates of the workshop are March 6-11, 2016. It will be held at Khon Kaen University (KKU) and Khon Kaen Zoo (KKZ), Khon Kaen, Thailand.

Program of the 7th ART Workshop 2016

6-Mar

Participants arrive Khon Kaen

7-Mar

Scientific forum at Faculty of Veterinary Medicine, Khon Kaen University

9.00-16.30

8-Mar

Workshop at Khon Kaen Zoo Welcome address: Tanachon Kensingh (Director of KKZ)

Overview of ART Workshop: Dr. Ampika Thongphakdee

Semen collection and cryopreservation

8.00-16.30 2 Thamin Eld's deer and 2 Hog Deer

Embryo collection and cryopreservation

9-Mar 4 Rusa deer 8.00-16.30

Embryo collection and cryopreservation 10-Mar 4 Rusa deer

8.00-16.30

11-Mar Conclusion

8.00-12.00


Agenda of the 7th Wildlife ART Workshop: March 7, 2016 at Faculty of Veterinary Medicine, Khon Kaen University

Time Topic Speaker

8.00-9.00 Registration

9.00-9.15

Welcome address Opening remarks

Assoc. Prof. Dr. Chuchart Kamollerd (Dean of Khon Kaen University, KKU) Benjapon Nakprasert (Director of Zoological Park Organization, ZPO)

9.15-10.00 Special lecture: Reproductive biotechnology: basic to application

Prof.Dr.Mongkol Techakumphu (Vice President of Chulalongkorn University, CU)

10.00-10.15 Group photo & coffee break

10.15-10.45 Why conservation and Wildlife ART in Thailand Sumate Kamolnorranart (DVM) (ZPO)

10.45-11.10 Health practice for captive breeding management Visit Arsaitumkul (DVM) (ZPO)

11.10-11.30 Best practice for wildlife anesthesia and reproductive case Chavin Chaisongkram (DVM) (KKZ, ZPO)

11.30-12.00 The role of oxidative stress in reproduction Dr.Yoswaris Semaming (DVM) (Udon Thani Rajabhat University)

12.00-13.00 Lunch

13.00-13.30 Cryopreservation of gametes and embryos: fundamental aspects Asst Prof. Dr. Theerawat Tharasanit (DVM) (CU)

13.30-14.00 Cryopreservation of sambar deer semen Assoc Prof. Dr. Thevin Vongpralub (KKU)

14.00-14.30 Semen evaluation and improvement of semen quality Asst Prof. Sarawut Sringam (DVM) (KKU)

14.30-14.50 Coffee break

14.50-15.20 Preliminary study on superovulation in rusa deer using a split-single intramuscular administration of follicle-stimulating hormone

Asst. Prof. Dr. Vibuntita Chankitisakul (DVM) (KKU)

15.20-15.50 Embryo development and quality assessment Prof. Dr. Yan-Der Hsuuw (DVM) (National Pingtung University of Science and Technology)

15.50-16.20 Embryo cryopreservation: slow freezing aspect Dr. Saksiri Sirisathien (DVM) (KKU)

16.30-16.40 Closing scientific session

16.40-17.10 Introduction for workshop (only participants for workshop)

18.00-20.00 Welcome dinner and transport to Khon Kaen Zoo

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Special Lecture:

Reproductive Biotechnology: Basic to Application

Mongkol Techakumphu

Department of Obstetrics Gynaecology and Reproduction, Faculty of Veterinary Science,

Chulalongkorn University, Bangkok, Thailand, 10300

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Why Conservation and Wildlife ART in Thailand

Sumate Kamolnorranart

Bureau of Conservation and Research, the Zoological Park Organization, Thailand

Increasingly altered and fragmented habitats directly affected natural wildlife

populations. More species are actively managed for their long-term survival. Conservation

breeding of endangered species is one of the crucial missions at the ZPO. Today, total of 532

species (10,280 individuals); 3,971 individuals of mammal, 4,961 individuals of birds, 1,329

individuals of reptiles and 19 individuals amphibians are cared in ZPO zoos. Seven member

zoos under the Zoological Park Organization (ZPO) are implementing ‘UN Decade on

Biodiversity’ campaign by contributing to specific strategies including; 1) maintaining genetic

diversity of captive wildlife population of global importance; 2) strengthen health

management, research programs on reproductive science and establish genome bank; and

3) reintroduction programs for the ‘extinct-in-the-wild’ species. Prioritized species selected

for IMP program are highlighted on Thai native endangered species such as Eld’s deer

(Rucervus eldii), Eastern Sarus crane (Grus antigone sharpii), Malayan tapir (Tapirus indicus),

goral (Naemorhedus griseus) and wild cats. The presentation will give the information “why

conservation and biodiversity are important?” and “update assisted reproductive technology

in wildlife in Thailand”.

3


Health Practice for Captive Breeding Management

Visit Arsaithamkul

Bureau of Conservation and Research, the Zoological Park Organization, Thailand

Wildlife captive breeding is a part of wildlife conservation, wildlife health is crucial

for wildlife conservation. The vital role of play in conservation initiatives have to consider

management ex situ wild animal relate to in situ wild animal. Zoo veterinarians and zoo

animal managing staffs have been challenged to apply management, welfare and animal

health not only for individuals but have more in preventive and therapeutic medicine for

populations. Working group on each specific species have to design as Scientific Advisory

Groups (SAGs) which comprised of zoo professionals and outside experts who research

specific topics ex. behavior and husbandry, reintroduction, veterinary science,

contraception, nutrition, small population management and genome resource banking, etc.

and establishing Species Survival Plan (SSP) programmes which is a co-operative population

management and conservation programme to facilitate the maintenance of a genetically-

viable and demographically-stable population of a specific species in captivity and educate

the public and foster basic veterinary, nutrition and reproductive research.

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Best Practice for Wildlife Anesthesia and Reproductive Case

Chavin Chaisongkram

Department of Research, Conservation and Animal Health, Khon Kaen Zoo, Khon Kaen,

Thailand, 40002

5


The Role of Oxidative Stress in Reproduction

Yoswaris Semaming

Veterinary Technology Program, Faculty of Technology, Udon Thani Rajabhat University,

Udon Thani, Thailand 41000

Free radical has been shown to play an important role in pathophysiology of various

degenerative diseases. Reactive oxygen species (ROS) are unstable and highly reactive.

They acquire electron from cells to become stable which resulting in cellular damage. Under

normal condition, scavenging molecules are known as antioxidants convert ROS to H2O

to prevent over production of ROS. There are two systems which consist of enzyme and

non-enzymatic antioxidants. Oxidative stress (OS) is the result of imbalance between ROS

and antioxidants which can lead to oxidative damage. In reproduction, oxidative stress has

been considered a major contributory factor to the infertility. Various factors including,

disease, pollutants, chemicals, radiation, pathogens, metabolic disorder and physical

disturbance promote excess free radical production. There are oxidative stress biomarkers

which could be detected in certain sample type both in vitro and in vivo (blood, cell, urine,

tissue and culture media) such as malondialdehyde (MDA), protein carbonyl content (PCC),

8-hydroxydeoxyguanosine (8-OHdG), nitric oxide, total antioxidant capacity, catalase,

glutathione and superoxide dismutase. Recently, oxidative stress has been identified as

important in assisted reproductive techniques (ART) including, intrauterine insemination,

in vitro fertilization, intracytoplasmic sperm injection. The impact of oxidative stress on ART

resulted in poor quality of oocyte and sperm and impaired embryonic development.

In order to promote assisted reproductive techniques success and improve reproductive

performance, the antioxidant supplementation should be considered.

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Cryopreservation of Gametes and Embryos: Fundamental Aspects

Theerawat Tharasanit

Department of Obstetrics Gynaecology and Reproduction, Faculty of Veterinary Science,

Chulalongkorn University, Bangkok, Thailand, 10300. Corresponding author Email:

[email protected]

Cryopresservation of gametes and embryos is a technique of choice for bio-banking

the genetic potential of desired animals. This technique allows long-term storage with

logistically acceptable if the cryopreserved materials would need to be transferred.

However, this technology frequently induces cryodamage that markedly affect cell viability

and functions. Several procedures during cryopreservation potentially induce cellular

changes that render poor cell viability and functions. Two cryopreservation techniques that

have been frequently used for cryopreservation of gametes and embryos are slow freezing

and vitrification. Slow cryopreservation requires an optimal freezing rate principally to

balance the intra- and extra-cellular water and cryoprotectant(s). In contrast to the slow

freezing technique, vitrification is a non-equilibrium cryopreservation technique that

requires high concentration of cryoprotectants and extremely fast freezing rate. Among

reproductive cells studied, sperm are the most successful cell types that well survive after

cryopreservation and thawing. However, successful cryopreservation is still problematic in

terms of sperm longevity and fertility post-thawing. It is well to note that sperm obtained

from different species differ in cryosensitivity; sperm of particular species would need to be

tested for cooling sensitivity prior to apply the technology to the field practice. Oocyte

cryopreservation is more challenging because overall success is currently poor.

The mammalian oocytes are the largest cells in the body that have low membrane

permeability to cryoprotectants and contain complexity of cell structures and organelles.

The dynamic changes of the oocytes contribute to high sensitivity of cryodamage. For

instance, the microtubules of meiotic spindle of mature oocytes are irreversibly damaged

during freezing and thawing processes. Embryos of particular species can also be

cryopreserved with variable success in terms of their viability and conception rates after

embryo transfer. Indeed, several factors have been demonstrated to affect cryosurvival of

embryos including stage of embryo development and culture condition. Stem cells derived

either from embryos or reprogramming of somatic cells have also been tested for feasibility

of genetic banking as they, in principle, have unlimitedly cell division and can differentiate

into specific cell types including gametes. However, the limitation of fundamental research

of this technology is lacking behind other simplified reproductive technologies.

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Cryopreservation of Sambar Deer Semen

Thevin Vongpralub1* and Wittaya Chinchiyanond2

1Department of Animal Science, Faculty of Agriculture, Khon Kaen University, 40002,

Khon Kaen, Thailand 2 Former Director of the Khao Kao Wildlife Breeding Research Center,

Petchaboon, Wildlife Conservation Bureau, National Park Wildlife and Plant Conservation,

Department Bangkok, 10900, Thailand *Corresponding author Email: [email protected]

ABSTRACT

Sambar deer (Cervus unicolor) are the large deer species native to India peninsula and

are found throughout Southeast Asia and Southern China. This species is listed as

vulnerable. Cryopreservation of semen and artificial insemination (AI) has played a major

role in propagation and conservation of genetic resources in wildlife species. For captive

populations frozen semen with AI has the potential for overcoming inbreeding depression

and maintaining genetic diversity. This review we describe the methods used for semen

cryopreservation and artificial insemination of deer with the main focus on main land

Sambar deer (Cervus unicolor equinus), with the hope of fostering future research and

fruitful area of Sambar deer reproductive technology.

INTRODUCTION

Sambar deer (Cervus unicolor) are the largest species of oriental deer and are found

throughout South Asia, Southeast Asia and Southern China. This species has been

introduced wildly outside it native range. Of the 14 subspecies described by Whitehead

(1993) Cervus unicolor equinus is the most prominent in Southeast Asia whereas Cervus

unicolor swinhoei is indigenous subspecies of sambar deer found in Taiwan. Wild

populations are rapidly declining in many Southeast Asian protected areas due to

commercial poaching (Leslie, 2011). In captivity, this cervid species has served as not only

recreational but also commercial farm deer in Chinese medicine enterprises. The used of

velvet antler in as an immune booster and as an antiallergy agent has increased in Taiwan

since the start of deer farming in 1963 (Kuo et al.,2012). Sambar deer is the preferred deer

meat in local and international markets (Steinmetze et al., 2006). This species of deer

attained slaughter weight earlier than red deer suggests that Sambar deer are worthy of

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domestication (Semiadi et al., 1995). Therefore, captive Sambar deer farming has become

commercially viable but good management requires a better understanding of the animal's

physiology. There is a general paucity of information on the reproductive physiology of

mainland Southeast Asian Sambar deer, both in the wild and in captivity.

As with domestic animals, cryopreservation of semen and artificial insemination (AI) has

played a major role in propagation and conservation of genetic resources in wildlife species.

For captive populations frozen semen with AI has the potential for overcoming inbreeding

depression and maintaining genetic diversity. A few studies to date have examined sperm

freezing and AI in the deer (Morrow et al., 2009).

Until recently, there has been only one study to determine the effect of different

diluents on post-thaw sperm quality in Formosan Sambar deer (Cervus unicolor swinhoie)

and Formosan Sika deer (Cervus nippon taiouanua). That study found that a different

diluent required different protocols for semen cryopreservation (Cheng et al. 2004). Since

specific protocols are required for optimal semen freezing for each breed or species. In the

past, adequate knowledge was still lacking on reproduction of Sambar deer in captivity.

During 2002 to 2004, we attempt to study the knowledge related to Sambar deer production

and research was carried out at the Khao Kao Wildlife Breeding Research Center,

Petchaboon, Thailand (16.66°N, 101.00°E, 700 m elevation). The practical protocols for

semen cryopreservation and AI in mainland Sambar deer (Cervus unicolor equinus) were

developed there. The intention of the following communication paper is to inform our

experience of AI technology in Sambar deer.

Semen production

Individual Sambar deer stags generally exhibit annual cyclicity in their antler cycles.

The males of this species exhibit alteration periods of fertility and infertility related to

dramatic changes in testis size and function. During antler regeneration, stag failed to show

sexual interest and gave poor semen quality (Somphol, 2004). During a period of winter and

early summer is a rust period, most of mature males have hardly antler and are highly

aggressive. According to our experience, high quality of semen for cryopreservation

mostly obtained during February-March. After a 65-75 days of growth period, velvet was

harvested. Stags were individually housed in outdoor pens (9 m x 12 m) and exposed to a

9


natural photoperiod and ambient temperature. Before semen collection period, mature

stags were hand-reared for gentleness in preparation for electro-ejaculation procedures.

Semen collection and evaluation

Semen was collected between the period in which stags exhibited reach maximum

breeding behavior. According to sedate procedure for deer was prohibited at wildlife

breeding research center, stags were manually restrained by experienced herdsmen under

intensive care observation. The electro-ejaculator was an Electrojac IV (Gemini Inc.,MN,

USA) with a rectal probe 2.5 cm in diameter and 20.5 cm in length with three longitudinal

electrodes. The electrical current intensity, from 0 to 12V, was divided in a 0 to 30 scale.

Electrical stimuli began at a low voltage and was gradually increased until semen was

collected. Stimuli and rest periods were two seconds each. During 2002-2004, more than

120 semen ejaculations were collected by this method with none of abnormal sign had been

observed in the donor stags. Chang et al.(2004) collected Formosan Sambar deer semen by

the similar method of animal control and reported that no injury was observed and

ejaculates were obtained with high success with the current electro-ejaculation approach.

Quality of raw semen was assessed by volume, concentration, forward progressive motility

(FPM) and normal sperm morphology (Evans and Maxwell, 1987). Only the semen with at

least 60 % FPM and 70 % normal sperm morphology was used in the experiments. The

cryopreserved semen evaluation included FPM and acrosome integrity as described by Dott

and Foster (1972).

Frozen semen processing

Beneficial of removal of seminal plasma on post-thaw semen quality

Removal of seminal plasma is recommended in goats (Ritar and Salamon, 1982), cattle

(AI-Somai et al., 1994) and buffalo (Ahmad et al., 1996). Sahni and Mohan (1990) reported

that toxicity of seminal plasma was higher among buffalo than cattle, which in turn

depended on the amount of seminal plasma. Maxwell et al. (2007) reported that, depending

on species, seminal plasma can either inhibit or stimulate sperm function and fertility. For

Iberian red deer Martinez-Postor et al. (2009) stated that seminal plasma might be

detrimental to cryopreserved sperm. In contrast, for Formosan Sambar deer, Cheng et al.

(2004) reported no negative effects of seminal plasma on the post-thaw quality.

10


Sperm freezability of the whole semen was lower than that of the seminal plasma

eliminated and additional of egg yolk diluent enhanced the quality of sperm collected by

electro-ejaculation. Notwithstanding, our study on liquid stored semen of Sambar deer

revealed that seminal plasma decreased the quality of diluted semen.

In our study, when seminal plasma was removed prior to redilution with tris-egg yolk

extender and processed for cryopreservation, the post-thaw quality was superior to aliquots

frozen in the present of seminal plasma (Vongpralub et al., 2015).

Semen freezing extender for Sambar deer

The effectiveness of extenders has been different between cervid species (Asher et

al., 2000; Chang et al, 2004; Jabbour et al., 1997). Most diluents used successfully in deer

have been adapted from sheep and goats (Asher et al., 2000). Of the various diluents used

for cryopreservation of deer semen, sodium citrate-egg-yolk-glyerol (Krzywinski and

Jaezewky, 1978) and Tris-glucose-citrate-egg yolk-glycerol (Evans and Maxwell, 1987) have

been usedc the most. Contributing to the body of knowledge, Cheng et al. [2004] compared

the efficacy of five extenders in cryopreservation of semen from Formosan Sika and Sambar

deer. Their results showed that Tris-Tes-egg yolk-glycerol extender for Eld’s deer and pig

frozen semen afforded better protection for Formosan Sika deer semen than the other

extenders. They also showed that Tris-citric acid- egg yolk-glycerol-based extender and an

extender for dog frozen semen and the previous extender were similar in their ability to

cryopreserve semen of Formosan Sambar deer.

A comparison of four egg yolk extenders (20%) included (1) Tris-egg yolk, (2) egg yolk

citrate, (3) Illinois Variable Temperature (IVT) extender (Salisbuly et al., 1978) and (4)

Beltsville F5 extender (Pursel and Johnson, 1975) was performed. For Thai Sambar deer

semen, Tris-egg yolk extender resulted in the highest post-thaw sperm progressive motility

rates compared to egg yolk citrate, Illinois Variable Temperature (IVT) and Beltsville F5

extenders. Also, the use of Tris-egg yolk extender resulted in the greatest acrosome

integrity (Vongpralub et al., 2015).

Optimal level of egg yolk concentration of the extender

Semen preservation techniques for cervine species have mostly been learned from

experience with sheep and goats. For Thai Sambar deer, no work has been reported on the

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optimal level of egg-yolk extender. Hen egg yolk is widely used as a cryoprotective agent in

semen freezing extenders in order to protect the spermatozoa against cold shock. The level

of egg yolk in extended semen is species dependent. In bovines (Vishwanath and Shannon,

2000) and porcines (Paquignon, 1985), about 20 % egg yolk has been commonly used in

frozen semen. On the other hand, concentrations of egg yolk above 2 % have proved

problematic in goats (Ritar and Salamon, 1982). But for deer semen, there are no reported

detrimental effects of egg yolk on semen quality when diluted with 2.5 to 20 % egg yolk

(Asher et al., 2000). In their study of five different extenders Cheng et al. [2004] used 20 %

egg yolk as part of the formulations for cryopreservation of Formosan Sambar deer semen.

For red deer, egg yolk extender was beneficial for semen preservation (Martinez-Postor et

al., 2009). For Thai Sambar deer semen, 20% (v/v) was the best concentration (P<0.05) of

Tris-egg yolk extender for freezing semen (Vongpralub et al., 2015).

Cooling rates on post-thaw semen quality

Cooling bovine spermatozoa from room temperature to 5 o C is generally done over a

period of 1.5 to 4 hr (Coulter and Foote, 1977; Foote and Kaproth, 2002; Rodriquez et al.,

1975; Senger et al., 1976; Wall and Foote, 1999; Wiggin and Almquist, 1975ab). Rapid

cooling to 5°C decreased the recovery rates of post-thaw spermatozoa (Dhami et al., 1992).

Prolonging the incubation and equilibration period before freezing also decreased the

survival rates (Pickett and Berndtson, 1974).

To our knowledge, for Thai Sambar deer, no previous studies have investigated

different cooling rates on the viability of frozen semen. There is also little information on the

optimal cooling methods in other cervids. Most reported procedures satisfactorily cooled

deer semen to 5 o C with equilibration periods modified from those for domestic ungulates.

Soler et al. (2003) reported that diluted semen of mature red deer, Père David’s deer and

fallow deer stags were refrigerated at 5 o C for 4 hr before freezing in liquid nitrogen vapour.

For Formosan Sika deer and Formosan Sambar deer, diluted semen was loaded into 0.25 ml

straws, slowly cooled to 5 o C in 2.5 hr and subsequently frozen in a programmable freezer

(Cheng et al., 2004).

To evaluate the effect of cooling rate on post-thaw survival of spermatozoa, semen

cooling rate, from 35 o C to 5 o C was accomplished over 1, 2, 3 or 4 hours. Semen samples

12


were cooled by dropping of ice cubes manually into ice bath. The cooling rates were

approximately 0.41, 0.21, 0.14 and 0.10o C/min respectively.

For Thai Sambar deer semen, 3 hours was the most beneficial cooling time (P<0.05)

from 35 o C to 5 o C prior to freezing (Vongpralub et al., 2015).

Glycerol concentrations on post-thaw semen quality

The discovery of glycerol as a cryoprotectant marked a quantum advance in semen

cryopreservation; however, a high level of glycerol can be detrimental to spermatozoa and it

is worth noting that inter-species variation in glycerol tolerance can be very marked (Holt,

2000). Loss of fertility was found when the concentration of glycerol exceeded 3% in boars

(Watson, 1979) , whereas the concentration in bull frozen semen ranged between 7% and

13%, depending on the extenders used (Salisbury et al., 1978). Rodriguez et al. (1975)

suggested that fast freezing resulted in a post-thaw motility equal to or greater than that of

the slower methods when the spermatozoa were suspended between 5% and 9% glycerol,

but the slower rates of freezing were superior when 11% glycerol was used. Matinez-Pastor

et al. (2009) found that 4% glycerol gave a better result for post-thaw quality of red deer

frozen semen, whereas Nally et al. (2011) reported that there was no difference when even

10% to 14% glycerol was used in Timor deer. In red deer, 4% to 6% glycerol has been used

for freezing semen (Garde et al., 2006). As reported by Cheng et al. (2004), glycerol

concentrations in successive extender used for semen cryopreservation of Formosan sambar

deer ranged between 5-8%. In addition, Lin et al.(2014) report that extender supplemented

with 6% glycerol was better for sperm viability, acrosomal integrity and motility in Formosan

Sambar deer.

For Thai Sambar deer semen, 3% was the best concentration (P<0.05) for freezing

semen compared to 5%, 7% and 9% glycerol (v/v) (Vongpralub et al., 2015).

Freezing rates on the post-thaw semen quality

No studies to date have examined the influence of different freezing rates on post-

thaw viability of Sambar deer spermatozoa. In cervines, the freezing methods have been

developed from ruminant species. In the freezing procedure for bull spermatozoa, semen

straws are generally held in static nitrogen vapor 2 to 4 cm above the liquid nitrogen or

at -100 o C to -160 o C (Landa and Almquis, 1979; Dhami et al., 1992; Johnson et al., 1995;

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Verma et al., 1994). The temperature at which the semen straw is held before immersing it

into the liquid nitrogen is between -80 o C and -150 o C(Jabbour et al., 1993; Garde et al.,

1998; Asher et al., 2000; Soler et al., 2003).

To evaluate different freezing rates on frozen thaw semen quality, the straws were

divided to three groups and maintained horizontally in the liquid nitrogen vapor at 2, 4 and 6

cm above the liquid nitrogen for 15 min before plunging into the liquid nitrogen. For Thai

Sambar deer semen, 4 cm was the best height (P<0.05) to suspend semen straws in the

liquid nitrogen vapor before plunging into the liquid nitrogen. Estimate of cooling rate were

12, 10, and 5o C/min for 2,4,and 6 cm heights respectively. Final temperature of cooling rate

were -180o C, 150o C, and -100o C respectively (Vongpralub et al., 2015).

Thawing temperature and time on post-thaw semen quality

In previous studies with cervid species, thawing temperature generally varied between

35 o C and 70 o C for between 8 and 60 sec. (Haigh et al., 1993; Monfort et al., 1993

Krogenase et al., 1994; Zomborszky et al., 1999; Hishinuma et al., 2003). Soler et al. (2003b)

compared the effect of thawing at 70 o C for 5sec, 50 o C for 8 sec and 37 o C for10 sec and

reported that the second and third protocols were the most beneficial for frozen semen

among the different cervid species. Although spermatozoa from Sambar deer were

cryopreserved and the thawing procedure was conducted at 37 o C (Cheng et al., 2004), the

effect of this procedure on semen quality has not been compared until now. The freezing

rates in this study can be considered rapid freezing. It has been argued that thawing should

also be done rapidly in order to avoid the re-crystallisation of large intracellular ice crystals

that can be harmful to the cell (Mazur, 1984).

For Thai Sambar deer semen, an intermediate thawing rate (50 o C for 8 sec) was better

than the slow (37 o C for 10 sec) or fast (70 o C for 5 sec) thawing rates (P<0.05) (Vongpralub

et al., 2015).

Freezing equipment

Diluted semen was loaded into 0.5 ml straws and held at 5 o C for few hr for

equilibration and then frozen in static liquid nitrogen vapor. Semen was frozen in styrofoam

boxes (25.5 x 37.5 x 29.0 cm) containing a 10 cm of liquid nitrogen. The temperature was

measured by TC 200® (Dickson, Il, USA), a digital thermometer.

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Artificial insemination

Sambar deer hinds estrous cycle

In Thailand, female Sambar deer (Cervus unicolor equinus) estrus is not seasonal,

normally 19 to 21 days estrous cycle and a gestation of 265.4 days are found

(Chinchiyanoon, personal communication (n=30)). In comparison, the estrous cycle and

gestation of female Formosan Sambar deer (Cervus unicolor swinhoei) were 18.2 and 258

days, respectively (Chan et al., 2009)

Estrous synchronization

To our knowledge, there is no published information on estrus synchronization and

timed AI in Sambar deer. Previous studies on other cervids have resulted in variable

behavioral estrus rates following various methods of estrous synchronization. Fallow deer

exhibited 50% to 100% estrus rates at 48 hr following CIDR-G (with PG) removal. Behavioral

estrus rates depended on the treatment duration, increasing from 50% for 8 days CIDR-G

insertion to over 90% for periods of at least 14 days (Morrow et al., 1995). In sika deer, a 13

day CIDR-G treatment followed by 50 IU of eCG at time of CIDR-G removal resulted in 36.6

2.9 hr to the onset of estrus (Willard et al., 1996). In most cases of estrus synchronized

farmed deer, the timing of AI is performed from 44 to 75 hr post-synchronization, depending

on the species (Willard et al., 2002). In induced estrus of red deer hinds with 150 IU of eCG

following CIDR removal, 96% were observed in estrus ranging from 29 to 91 hr (mean 47.5 ±

3.3 hr) (Bowers et al., 2004). In Eld’s deer (Cervus eldi thamin), after 14 days of CIDR-G

insertion, behavioral estrus was detected in 12 of 20 (60%) hinds (Monfort et al., 1993).

In our study (Vongpralub et al., 2015), estrus was synchronized on ten hinds by using

13 days of the controlled progesterone releasing intra-vaginal device for goats (CIDR-G)

(Inter Ag, Hamilton, New Zealand) followed at the time of withdrawal by IM injection of 200

IU equine Chorionic Gonadotropin (eCG) (Folligon; Intervet International BV, Holland) and

PGF2α (375 µg cloprostenol; 1.5 ml estroplan; Parnell laboratories Pty, Ltd, Australia).

Protocol for timed insemination was determined by preliminary estrus synchronization

studies (n=8) in which estrus was detected by closely observing the behavior of a trained,

mature stag as he interacted with hinds in 12 hr intervals. Onset of estrus was determined

when the hinds allowed a 5 minute one-to-one interaction with the stag. Estrus was

observed 30 hr after CIDR-G withdrawal and lasted 24 to 60 hr.

15


AI with frozen semen in Sambar deer

In our study, the highest quality semen from one stag was used to inseminate ten

hinds. Quality was determined by semen volume, percentage of motile sperm, percentage

of live sperm, percentage of normal sperm, and sperm concentration which was 1 ml, 85%,

95%, 91% and 1,400 million cells/ml, respectively. The best methods, as determined from

the results of above experiments, were used to freeze semen with 20 x 106 cells/straw.

Progressively motile sperm of the thawed semen was always more than 65 % motility.

To achieve insemination a bovine breeding gun was inserted into a foam type catheter

for pig AI (pig champ). The modified catheter was then inserted through the vagina, close to

the cervix. Adjustments were then made to the tip of the catheter, and its correct position

in the cervix was confirmed by rectal examination, so that the bovine gun could be inserted

through the first ring of the cervix to deposit the semen. One time of AI, only one straw was

used for each female. According to sedated procedure was prohibited, the hinds were also

manually restrained with the soft net and under intensive care. All ten Sambar hinds (100%)

exhibited estrus at 28.8 ± 11.2 hr following removal of the CIDR-G and injections with 200 IU

eCG and PGF2α. The duration of estrus was 28.0 ± 6.1 hr. The hinds were manually restrained

and duration of restraint including insemination for each hind was less than 10 min. Artificial

insemination was performed in 2.8 ± 1.0 min. Pregnancy rates and fawns born were not

different when comparing single versus double timed AI (48 hr versus 48 hr + 12 hr,

respectively) following estrus synchronization (P>0.05). After 60 days, non-return to estrus

was 60% for both treatments. The fawning rate for single AI was 40% versus 60% for double

AI (P>0.05).

CONCLUSION

Since specific protocols are required for optimal semen freezing for each breed or

species. Little information has been reported regarding semen cryopreservation and AI

technology in Thai Sambar deer. Our studies demonstrated for the first time that the most

appropriate cryopreservation protocol for the highest percentage of viable/motile Sambar

deer semen (spermatazoa). (1) Discard the seminal plasma immediately after semen

collection. (2) Use Tris-egg yolk at 20% concentration with 3% glycerol for the most suitable

extender. (3) Cool from room temperature to 5 o C over a period of 3 hours. (4) Place the

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semen straws in the vapor 4 cm above the liquid nitrogen, before freezing, (5) Thaw the 0.5

ml frozen semen straws at 50o C for 8 sec for optimal motility.

Estrus was successfully synchronized in Thai Sambar deer using CIDR-G for 13 days

followed by 200 IU eCG and PGF2α. Using the high quality frozen semen, a single timed

insemination at 48 hr post-CIDR removal resulted in a accetable pregnancy rate of 60 %.

The knowledge gained from this study could serve as a useful model for cryopreservation

and AI in Cervus unicolor or other critically endangered deer species in captivity or under

field conditions.

ACKNOWLEDGMENTS

Appreciation is expressed to the National Park, Wildlife and Plant Conservation

Department for financial support. We also thank the staff of the Khao Kao Wildlife Breeding

Research Center for their assistance in all aspects of raising, handling behavioural

observation, and artificial insemination of the deer.

17


Semen Evaluation and Improvement of Semen Quality

Sarawut Sringam

Department of Veterinary Surgery and Theriogenology, Faculty of Veterinary Medicine,

Khon Kaen University, Thailand 40002. Corresponding author Email: [email protected]

Electroejaculation (EE) is the only technique useful for collecting good quality of

semen from wild animals, and the postmortem sperm recovery may be an alternative

technique in some situation. Semen consists of spermatozoa mixed with secretions from

testes, epididymides and accessory organs of the male reproductive system. The method to

evaluate semen fertilizing capability is through the results obtained by in vivo fertility.

However, as it is difficult to apply this method in practice, especially in wild animals. It is

agreed that spermatozoon is multifunctional cell that must possess a large number of

attributes that make it potentially fertile. A fertile ejaculate must meet certain semen

parameter quality standards (?), such as: progressive motility, normal morphology, active

energy metabolism, structural integrity and functionality of the membrane, penetration

capacity and optimum transfer of genetic material. The analysis of a single sperm parameter

cannot predict the outcome of a process as complex as fertilization. In order to increase the

predictive power of the test, simultaneous analysis of multiple sperm attributes should be

done.

Some of male animals cannot give the standard quality of semen. Semen collected by

EE is often more dilute and may appear to be less motile than that collected by artificial

vagina. Some different techniques are used to collect the spermatozoa before frozen or use

for artificial insemination, but the choice strongly depend on the quality of the semen that is

on the concentration, motility and morphology, in order to obtain the higher number of

good spermatozoa. The principle techniques of sperm preparation consist of washing,

migration and density gradient centrifugation techniques. In addition, the further studies

about extender and various antioxidants on frozen semen procedures will increase the

success in ART.

18


Preliminary Study on Superovulation in Rusa Deer using a Split-single

Intramuscular Administration of Follicle-stimulating Hormone

Vibuntita Chankitisakul1*, Ampika Thongphakdee2, Chavin Chaisongkram3,

Mongkol Techakumphu4

1Department of Animal Science, Faculty of Agriculture, Khon kaen University, Khon Kaen,

Thailand, 40002 2 Wildlife Reproductive Innovation Center, Bureau of Conservation and

Research, Zoological Park Organization, 71 Rama 5, Dusit Bangkok 10300 3 Department of

Research, Conservation and Animal Health, Khon Kaen Zoo, Khon Kaen, Thailand, 40002

4 Department of Obstetrics Gynaecology and Reproduction, Faculty of Veterinary Science,

Chulalongkorn University, Bangkok, Thailand, 10300

*Corresponding author Email: [email protected]

Cervids represent more than 200 subspecies divided amongst 17 genera,

characterized by striking differences in geographical distribution, morphology and

physiology. Within cervid subspecies, almost 40 are considered to be threatened with

extinction by the International Union for Conservation of Nature and Natural Resources

(IUCN Red List). In Thailand, Schomburgk’s Deer (Rucervus schomburgki) was extinct around

1932, with the last captive individual being killed in 1938. None of the indications that the

species inhabited other countries can be shown to have any compelling basis and some are

clearly in error. Thus the species is considered a single-country endemic, to Thailand, where

it is certainly extinct. Besides Schomburgk’s Deer, others wildlife species such as Eld’s deer

(two subspecies of Eld’s deer, thamin (Rucervus eldii thamin) and siamensis (Rucervus edlii

siamensis)) have been counted as reserved species under the Thai Wildlife Preservation and

Protection Act (1992) and are also classified as endangered species by IUCN Red List because

there are no appropriate conservation measures. Therefore, ex situ conservation programs

have been strongly proposed to prevent genetic loss and to increase the prolificacy of

remaining individuals. The application of biotechnologies developed for domestic ruminants,

such as superovulation and embryo transfer is a rapidly growing biotechnology, and had

already been successfully.

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Superovulation for induction of multiple follicular developments in embryo transfer

program normally requires administration of an exogenous gonadotropin preparation that

mimics the effect of FSH. The gonadotropin must be available long enough for the follicle to

grow and attain final maturation of the oocyte. The biological half-life of exogenous FSH

normally has a relatively short and multiple treatments are needed to induce

superstimulation. Generally FSH is injected intramuscularly twice daily over 3-5 day period.

The administration of FSH therefore requires constant attention by personnel, increasing the

possibilities of failures due to mishandling and errors with the administration. Especially in

case of wildlife animals, capture strategies for immobilization of wildlife is a procedural

stress of the operator and can cause undue stress and harmful to animals. Subsequently

suppressive to the superstimulatory response could possibly occur. Thus simplified protocols

for superstimulation of follicular development may be expected to reduce costs, handling

and improve response in deer. The development of a single dose protocol of FSH would be a

useful alternative to the traditional twice daily. A single injection of FSH was achieved by

diluting the hormone with a biodegradable polymer such as polyvinylpyrrolidone (PVP)

or/and a hyaluronan based slow-release formulation (SRF) that resulting in sustained, slow

release of the hormone over several days. To the best of my knowledge, few studies of

superovulation and embryo transfer in deer were reported, particularly only one study of

superovulation with a single dose of FSH injection was found unsuccessfully in year 2012.

Therefore this study was conducted in order to develop an effective superovulatory protocol

for deer that minimizes animal handing. We hypothesized that the slow release FSH

provides the same ovarian response in superovulation program in rusa deer which would be

a new fundamental knowledge of superovulation and could improve assisted reproduction

technologies for other deer species in the future.

Materials and Methods

Rusa deer (n=13) were synchronized by inserting a controlled internal drug release

(CIDR; Pfizer, USA) for 12 days. The date of CIDR insertion is designated Day 0 (initial day of

treatment). Then they were assigned randomly into two treatments for superstimulation as

follows;

Treatment 1; Multiple injection treatment: Superovulation with multiple injection

was conducted with a total dose of 180 mg of NIH-FSH-P1 (Folltropin-V, Bioniche,

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Belleville,ON, Canada). Intramuscular injections are given every 12 h for 8 injections, (22.5

mg at each injection), beginning on the afternoon of Day 9. The CIDRs were removed from

donors on Day 12, concurrently with the 7th injection of FSH.

Treatment 2; Split-single dose treatment: Superovulation with slow release FSH

injection: The slow-release FSH treatment was prepared in a diluent (MAP-5 50 mg) and

given split into two; 120 mg of FSH was administered intramuscular on the afternoon of Day

9 and the remaining 60 mg of FSH was administered 48 hours later. The CIDRs were

removed from donors on Day 12.

The number of corpus luteum were recorded and compared between two

superovulation treatments by laparoscopy.

Results and Discussion

The mean numbers of corpus luteum (Fig.1A) were higher in the multiple injection

treatment compared with split-single injection treatment (4.25 vs. 2.5) (P<0.05). In addition,

we noticed that the ovaries which unresponsive to gonadotropins had smaller size (Fig.1B)

compared with the others (P<0.05). However, we could not make the summary at the

moment because this study was only preliminary study. The results of hormonal profiles and

more sample size are needed to conduct further. The preliminary result provides the

primary information that is possible to apply the biotechnology to other endangered deer

species in the future.

Figure.1 the corpus luteum on ovary (A); the inactive ovary (B)

(A) (B)

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Acknowledgements

This study was financially supported by the Thailand Research Fund and the

Zoological Park Organization. We are grateful to Asst.Prof.Dr.Wuttigrai Boonkum for his

assistance in statistical analysis. We would like to thanks for Dr. Yoswaris Semaming,

Dr. Kanda Ponsrila and Dr. Aunchisa Phojun for their surgical assistance and anesthesia

procedure during the experiments. Thank you to keepers of Khon Kaen zoo and technicians

of the Wildlife Reproductive Innovation Center for their kind attempt on animal care and

collaboration.

22


Embryo Development and Quality Assessment

Yan-Der Hsuuw

Department of Biological Science and Technology

Director, Laboratory Animal Center, Embryo development and Stem Cell Laboratory,

National Pingtung University of Science and Technology, Neipu, Pingtung, 912 Taiwan

The development of embryo is critically controlled by a precise cooperation of

hormones and regulators (cytokines and growth factors) present in the reproductive tract.

Learn more about the biological effects of hormones or regulators on the preimplantation

embryo development, implantation as well as on embryogenesis and pregnancy

maintenance, could therefore provide and important trail to understand the interactions

between maternal receptivity and embryo viability during pregnancy. To evaluate the

viability of preimplantation embryo in vitro, we have performed several methods on

embryonic cell growth, differentiation or cell death at the time or after implantation.

Differential staining, is based on immunosurgery, considering the impermeability of the

trophectoderm (TE) layer which protects the inner cell mass (ICM) from the exposure to the

antibody and complement reaction. Two cell lineages can be distinguished following the

dual fluorochromes staining, bisbenzimide (Hochest 33258) and propidium iodide (PI). Cells

containing fragmented nuclei (karyorrhexix) are identified as dead cells. Outgrowth assay,

the ability of blastocyst to implant and develop can be assessed in the light of an outgrowing

culture model. The hatched blastocysts will attach onto the fibronectin and outgrow with a

cluster of ICM over the TE outgrowth. The proliferation of outgrowths is examined by the

counting the nuclei directly on the dish following the BrdU incorporation and the cell

spreading technique. TUNEL assay, Terminal transferase-mediated dUTP nick end labeling is

based on the binding of dUPT to each 3’-hydroxyl terminal of DNA strands. Cells containing

fragmented DNA (karyolysis) is identified as apoptosis. In conclusion, a sufficient number of

ICM cells are imperative in the process of implantation, and the excessive reduction in this

cell lineage may impair embryo viability in spite of a normal TE population or a normal

implantation rate. Monitoring the embryo quality by different approaches will be helpful to

estimate the optimum system in embryo culture, and to promote a successful pregnancy

following the embryo transfer.

23


Improvement in cryopreservation of in vitro produced bovine embryos

Saksiri Sirisathien

Dept. of Surgery and Theriogenology, Fact. of Vet. Med., Khon-Kaen University

Cryopreservation of embryos has become an essential part of assisted reproductive

technology (ART) allowing embryos to be used with its full potential. Cryopreservation of in

vivo derived bovine embryos is now a routine procedure in embryo transfer program.

However, in vitro produced (IVP) bovine embryos differ from the vivo derived embryos in

several aspects, especially its cryotolerance. The cryopreservation of IVP bovine embryos

remains to be improved even though it was established for decades. Experiments were

conducted to improve slow freezing technique for IVP bovine embryos using simple

empirical approach. In experiment 1, effect of different base media and types of

cryoprotective agents were examined. Bovine expanded blastocysts (8 days after

fertilization) were cryopreserved in the freezing media consisted of 10 % ethylene glycol

(EG) alone or 5% EG + 5% dimethyl sulfoxide (DMSO) diluted in one of the three base media

(TCM-199, D-PBS, or cytomix) plus 0.1 M sucrose and 10% bovine serum. The initial survival

rate at 24 h post thawed of embryos cryopreserved with 5% EG + 5% DMSO in three base

media combined was higher than that of embryos cryopreserved with 10% EG alone (87.1%

vs. 71.4%, respectively, p< 0.05). No effect of base media on the cryosurvival of embryos

was detected. In experiment 2, embryos were cryopreserved with 5% EG+ 5% DMSO in

D-PBS. The temperature was cooled down to reach -30 or -40ºC before plunged into liquid

nitrogen. The plunging temperature had no effect on the initial survival rate at 24 h post

thawed. However, hatching rate at 72h post thawed was higher in -40ºC group compared to

that of -30ºC group (70.6% vs. 39.4%, respectively). In experiment 3, embryos were

cryopreserved with 0.1 M sucrose or 0.1 M trehalose or 0.1 M raffinose. Replacing sucrose

with trehalose or raffinose had no effect on cryosurvaival of embryos. In conclusion, a slow

freezing technique using 5% EG + 5% DMSO in D-PBS plus 0.1 M sucrose and 10% bovine

serum with the plunging temperature at -40ºC was found to be an efficient procedure for

cryopreservation of IVP bovine embryos.

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Documents

Proceedings of The 7 Wildlife Assisted Reproductive ... · “The 7th Wildlife Assisted Reproductive Technology (ART) Workshop” This workshop is aimed to establish one of the stages