Sustainable Livestock Production in the Perspective of Food Security, Policy, Genetic Resources, and Climate Change

  P A A A A A A P

  The 16 _{AAAP th} Sustainable Livestock Production in the Perspective of _Congress Food Security, Policy, Genetic Resources, and Climate Change Food Security Sustainable Livestock Production in the Perspective of

  Proceedings , Policy

  Full Papers , Genetic Resources, and Climate Change

  Proceedings Full Papers th

  The 16 AAAP Congress

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   Scope of AAAP: AAAP is established to devote for the efficient animal production in the Asian-Australasian region through national, regional, international cooperation and academic conferences.

   Brief History of AAAP: AAAP was founded in 1980 with 8 charter members representing 8 countries-those are Australia, Indonesia, Japan, Korea, Malaysia, New Zealand, Philippines and Thailand. Then, the society representing Taiwan joined AAAP in 1982 followed by Bangladesh in 1987, Papua New Guinea in 1990, India and Vietnam in 1992, Mongolia, Nepal and Pakistan in 1994, Iran in 2002, Sri Lanka and China in 2006 , thereafter currently 19 members.

   Major Activities of AAAP: Biennial AAAP Animal Science Congress, Publications of the Asian-Australasian Journal of Animal Sciences and proceedings of the AAAP congress and symposia and Acknowledgement awards for the contribution of AAAP scientists.

   Organization of AAAP:

  ∙ President: Recommended by the national society hosting the next biennial AAAP Animal Science Congress and approved by Council meeting and serve 2 years. ∙ Two Vice Presidents: One represents the present host society and the other represents next host society of the very next AAAP Animal Science Congress. ∙ Secretary General: All managerial works for AAAP with 6 years term by approval by the council ∙ Council Members: AAAP president, vice presidents, secretary general and each presidents or representative of each member society are members of the council. The council decides congress venue and many important agenda of AAAP  Office of AAAP: Decided by the council to have the permanent office of AAAP in Korea.

  Currently # 909 Korea Sci &Tech Center Seoul 135-703, Korea  Official Journal of AAAP: Asian-Australasian Journal of Animal Sciences (Asian-Aust. J. Anim. Sci. ISSN 1011-2367. http://www.ajas.info ) is published monthly with its main office in Korea  Current 19 Member Societies of AAAP:

  

ASAP(Australia), BAHA(Bangladesh), CAASVM(China), IAAP(India), ISAS(Indonesia),

  

IAAS(Iran), JSAS(Japan), KSAST(Korea), MSAP(Malaysia), MLSBA(Mongolia),

NASA(Nepal), NZSAP(New Zealand), PAHA(Pakistan), PNGSA(Papua New Guinea),

PSAS(Philippines), SLAAP(Sri Lanka), CSAS(Taiwan), AHAT(Thailand), AHAV(Vietnam).

   Previous Venues of AAAP Animal Science Congress and AAAP Presidents

  I 1980 Malaysia S. Jalaludin

  II 1982 Philippines

  V. G. Arganosa

  III 1985 Korea In Kyu Han

  IV 1987 New Zealand

  A. R. Sykes V 1990 Taiwan T. P. Yeh

  VI 1992 Thailand

  C. Chantalakhana

  VII 1994 Indonesia

  E. Soetirto

  VIII 1996 Japan T. Morichi

  IX 2000 Australia J. Ternouth X 2002 India P. N. Bhat

  XI 2004 Malaysia Z. A. Jelan

  XII 2006 Korea

  I. K. Paik

  XIII 2008 Vietnam N.V. Thien

  XIV 2010 Taiwan L.C. Hsia

  XV 2012 Thailand C.Kittayachaweng XVI 2014 Indonesia Yudi.Guntara.Noor

  _{10-14 November 2014, Gadjah Mada University, Yogyakarta, Indonesia Proceedings of the 16 AAAP Animal Science Congress Vol. II} th

  

CONTENTS

ORAL PRESENTATION Code Title Page Genetic and Reproduction

  Large Ruminants

  

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   POSTER PRESENTATION Code Title Page Genetic and Reproduction

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Effects of Diluents and Cryoprotectants on Native Chicken Sperm Motility

W. Asmarawati, Kustono, D. T. Widayati, S. Bintara and Ismaya

  Faculty of Animal Science, Gadjah Mada University Yogyakarta, Yogyakarta, Indonesia Corresponding email: widyasmarawati@gmail.com

  ABSTRACT

  This research conducted to determine the influence of various cryoprotectants and diluents on post-thawing semen quality. Two diluent used were buffer phosphate (BP) and egg-yolk buffer phosphate (EYBP). Three cryoprotectants added to the diluents were glycerol (G), dimethyl formamide (DMF) and dimethyl sulfoxide (DMSO). Semen were collected from 4 males chicken aged 1 year to 1.5 years. After examined, 2 different diluents and 3 different cryoprotectants were adding into polled semen that was devided into 6 different treatment tubes (BP+G 7%, BP+DMF7%, BP+DMSO7%, EYBP+G 7%, EYBP+DMF7%, and EYBP+DMSO7%), then prosessed to be frozen with slow freezing method. Motility before dan after equilibration were examined. Frozen semen was stored at temperature -196 °C for a week, then thawed and its motility were examined. Fresh sperm quality were analyzed with mean and standard deviation, whereas data of motility before equilibration, after equilibration, and post-thawing were analyzed by analysis of variance using completely randomized design factorial 2 x 3. The result showed that diluents treatment gave significant differences (P ≤ 0.05) on sperm motility after equilibration and post-thawing, but not gave significant differences on sperm motility before equilibration. Cryoprotectant treatments gave significant differences (P ≤ 0.01) on sperm motility before equilibration and after equilibration, but not gave significant differences on post-thawing sperm motility. It can be concluded that DMF adding into EYBP diluents gave highest post-thawing sperm motility.

  Key Words: Native chicken, Diluent, Cryoprotectant, Sperm motility.

  INTRODUCTION

  Various methods and procedures for freezing chicken semen have been studied for many years. Technology of artificial insemination face the major problem that is declining of sperm motility just in a few moment after ejaculation andstart losing ability to fertilize (Tri- Yuwanta et al., 1998). Cryopreservation can be defined as problem solving for future use, cryopreservation of cock semen remains a challenging obstacle due to low survival and motility rate of frozen-thawed cock semen (Makhafola et al. 2009). Fowl sperm have low resistance to the formation of internal ice crystals and reduce (Seigneurin and Blesbois, 1995), which reduce viability and motility. Motility is used as the most important value to measure sperm fertilizing ability. The critical steps in fowl semen cryopreservation are selection of suitable semen extender, proper cryoprotectant, and thawing method (Suidzinska and Lucaszewics, 2008; Makhafola et al., 2009). Egg-yolk buffer phosphate diluent is a balanced blending of phosphate buffer solution and fresh egg yolk, this diluent maintaining sperm motility and fertility (Bearden and Fuquay, 1997). The addition of glycerol as a cryoprotectant agent in frozen chicken semen as much as 8%, 12%, and 16% gave post- thawing sperm motility 48%, 50%, and 52% respectively (Saleh and Sugiyatno, 2007). Setioko et al. (2002) useglycerol, dimethyl sulfoxide (DMSO) and dimethyl formamide (DMF) in freezing duck semen with the results of post-thawing sperm motility 9.02%; 21.75%; and 32.86% respectively.The purpose of this study was to determine and comparing the effects of twodiluent used for native chicken frozen semen i.e.buffer phosphate (BP) and egg-yolk buffer phosphate (EYBP). Also, combine with three kind of cryoprotectants i.e glycerol (G), dimethyl formamide (DMF) and dimethyl sulfoxide (DMSO).

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  1RYHPEHU *DGMDK 0DGD 8QLYHUVLW\ <RJ\DNDUWD ,QGRQHVLD MATERIALS AND METHODS Materials of this research were semen from 4 males native chicken aged 1 year to 1.5 years.

  Males were housed in individual cages under natural light. Males were fed in ad libitum diet with commercial feed for native chicken R-19 produced by PT Japfa Comfeed Surabaya. The extenders for semen was Buffer phosphate (BP) and egg-yolk buffer phosphate (EYBP) that was buffer solution from Na-phosphate monobasis, Na-phosphate dibasis, aquadest, and gentamicine 0.5 mg/100 mL. Adding of chicken egg yolk to phosphate buffer was needed for making EYBP. Three cryoprotectants added in diluents were Glycerol (G), Dimethyl Formamide (DMF) and Dimethyl Sulfoxide (DMSO). Semen was collected by Burrows and Quinn method (1937) that is dorso-abdominal massage (Donoghue and Wishart, 2000).Fresh semen from 4 cockerels was pooled and assessed for semen quality, which include: volume, color, consistency, motility, concentration, viability, and abnormallity. Semen extender was used Buffer phosphate (BP) with Gentamicin 0.5 mg/100 mL. Diluent EYBP was made from BP supplemented with 20% fresh egg yolk. Two kinds of extenderdivided into six different tubes and added with three kinds of cryoprotectants. Six extender treatments were BP+G 7%, BP+DMF7%, BP+DMSO7%, EYBP+G 7%, EYBP+DMF7%, EYBP+DMSO7%, respectively. Pooled semen from 4 cockerels after assessed, then divided into six equal parts. Each parts was diluted into each extender treatment, with ratio of sperm and extenders was 1:

  2. Motility observations were carried out for three times i.e before equilibration,after equilibration and post-thawing. After motility observation, samples were equilibrated for 1 hour at 4 °C. After equilibration time, sperm motility were assessed. The semen samples were

  2

  directly filed into the straw and placed in pre-freezing unit (steam above LN for 8 minutes),

  2

  then directly in LN container for frozen storage for at least 1 week before being thawed for post-thawing motility assessed. The semen samples were thawed at temperature 37 ° C for 30 seconds. Sperm motility was evaluated by visual determination of sperm with progressive motions using microscope. Data obtained from fresh sperm (includes volume, color, consistency, motility, concentration, viability, and abnormallity) was analyzed with a mean and standard deviation. Sperm motility before equilibration,after equilibration and post- thawing were analyzed with analysis of variance using completely randomized design factorial 2 x 3 and followed by DMRT test(Astuti, 1980).

  RESULTS AND DISCUSSION Fresh Sperm of Native Chicken Fresh semen characteristic from four males of native chicken showed in Table 1 below.

  Table 1 . Fresh sperm characteristic of native chicken Parameter

  Result (average ± SD) Volume (mL) 0.66 ± 0.28 Colour White Consistency ₉ Viscous Concentration (x 10 spermatozoa/mL) 2.01 ± 0.42

  Motility (%) 81.43 ± 8.52 Viability (%) 84.77 ± 4.12 Abnormality (%) 16.42 ± 6.25

  The results indicates fresh sperm of native chicken had average of volume, colour, consistency, concentration, motility; viability,and abnormality. Results of this research within normal limits, which was volume ranges from 0.1 to 0.9 ml (Etches, 1996); viscous white

  9

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  color (Utomo, 1997; concentrations between 3.10 to 7.10 spermatozoa/mL (Ax et al., 2000); viabilityaverage around 96,64% and abnormality around 8% (Blesbois et al., 2005).

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  Sperm Motilities before Equilibration

  Sperm motilitiesbefore equilibration insix treatments of diluent BP+G 7%, BP+DMF7%, BP+DMSO7%, EYBP+G 7%, EYBP+DMF7%, EYBP+DMSO7% showed in Table 2 below.

  Table 2 .Sperm motilities before equilibration Cryoprotectant ns Extender average Glycerol 7% DMF 7% DMSO 7%

  BP 57,50 ± 2,89 68,75 ± 7,50 52,00 ± 2,45 59,42 ± 8,50 EYBP 50,00 ± 8,16 70,00 ± 4,08 51,25 ± 7,50 57,08 ± 11,37

_{a b a}

Average 53,75 ± 6,94 69,38 ± 5,63 51,63 ± 5,18 _{a, b ns} : Data with different superscripts within each row were significantly different (P≤0.01) : non significant

  The result showed that diluents treatment not gave significant differences on sperm motilities before equilibration. Cryoprotectant treatments gave significant differences (P ≤ 0.01) on sperm motility before equilibration. The sperm motility before equilibration in DMF 7% treatment was significantly higher than that obtained with glycerol or DMSO. It is not much different from the results obtained by Setioko et al. (2002) thatuse DMF 7% as cryoprotectants in the freezing duck semen provide motility 74.53% after dilution. Under this research condition, DMF 7% was the best cryoprotectant tested for native chicken semen motility before equilibration in BP and EYBP diluents. Observations also indicate that there was significant interaction between diluentsand cryoprotectants on sperm motility.

  Sperm Motilitiesafter Equilibration

  Sperm motilitiesafter equilibration insix treatments of diluentBP+G 7%, BP+DMF7%, BP+DMSO7%, EYBP+G 7%, EYBP+DMF7%, EYBP+DMSO7% showed in Table 3 below.

  Table 3 .Sperm motilities after equilibration Cryoprotectant

  Extender Average Glycerol 7% DMF 7% DMSO 7% _x BP 48,75 ± 2,50 63,75 ± 2,50 63,75 ± 2,50 58,75 ± 7,72 _y EYBP 43,75 ± 4,43 63,75 ± 9,46 50,00 ± 7,07 52,50 ± 10,98

_{a b b}

Average 46,25 ± 4,43 63,75 ± 6,41 56,88 ± 8,84 _{a, b x, y} : Data with different superscripts within each row were significantly different (P≤0.01) _ns : Data with different superscripts within each column were significantly different (P≤0.05) : non significant

  The result showed that diluents treatment gave significant differences (P ≤ 0.05) on sperm motilities after equilibration. The sperm motility in BP was significantly higher than EYBP. Cryoprotectant treatments gave significant differences (P ≤ 0.01) on sperm motilities after equilibration. The sperm motilities after equilibration in DMF 7% and DMSO 7%were significantly higher than obtained with glycerol.Similar result from Setioko et al. (2002), also use DMF 7% as cryoprotectants to freezing duck semenand gave sperm motility 63.64% after equilibration. Under this research condition, DMF 7% was the best cryoprotectant tested for native chicken semen motility after equilibration in BP and EYBP diluents. Observations also indicate that there was significant interaction between type of diluents and type of cryoprotectants in affecting sperm motility after equilibration.

  Post-thawing Sperm Motilities

  Sperm motilitiesafter thawing insix treatments of diluentBP+G 7%, BP+DMF7%, BP+DMSO7%, EYBP+G 7%, EYBP+DMF7%, EYBP+DMSO7% showed in Table 4 below. The result showed that diluents treatment gave significant differences (P ≤ 0.05) on post- thawingsperm motility. Post-thawing sperm motility in BP was significantly higher than that obtained in EYBP. Cryoprotectant treatments not gave significant differences on post-

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  thawing sperm motility.Post-thawing sperm motility in DMF 7% was higher than DMSO 7% and Glycerol 7%.

  Table 4.

  Post-thawing sperm motilities Cryoprotectant Extender

  Average Glycerol 7% DMF 7% DMSO 7% _x BP 25,00 ± 4,08 38,75 ± 8,54 26,25 ± 13,77 30,00 ± 10,87 _y EYBP 25,00 ± 5,77 25,00 ± 10,00 15,00 ± 7,07 21,67 ± 8,62 _{ns x, y} Average 25,00 ± 4,63 31,88 ± 11,32 20,63 ± 11,78 _ns : Data with different superscripts within each column were significantly different (P≤0.05) : non significant

  Makhafola et al. (2009) also use DMSO 5% as cryoprotectants to freezing semen from White Leghorn cockerel and gave sperm motility up to 40±4.7%; 20±4.0%; 15±3.7%; and 12±3.6% for 0 minute thawing; 30 minutes thawing; 60 minutes thawing; and 90 minutes thawing, respectively.Avian sperm are more sensitive to the freezing and thawing process and fertility rates of cryopreserved poultry sperm are dramatically lower than any of the domestic mammalian species (Donoghue and Wishart, 2000; Long, 2006; Gerzilov, 2010). Observations of post-thawing sperm motility also showed that there was no significant interaction between the type of diluentsto the type of cryoprotectants in affecting sperm motility.Under this research condition even though was not significantly, DMF 7% was the best cryoprotectant tested for post-thawing native chicken semen motility in BP and EYBP diluents.

  REFERENCES

  Astuti, J. M. 1980. Rancangan Percobaan dan Analisis Statistik, bagian I. Bagian Pemuliaan Ternak, Fakultas Peternakan UGM, Yogyakarta. Ax, R.L, M. Dally, B.A. Didion, R.W. Lenz, C.C. Love, D.D. Varner, B. Hafez, and M.E.

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  Bellin. 2000. Semen Evaluation. In: Reproduction in Farm Animals 7 ed. E. S. E Hafez. Ed., Lea & Febiger, Philadelphia. Pp. 365-375. Bearden, H. J., and J. W. Fuquay. 1997. Applied Animal reproduction: Fourth Edition.

  Prentice-Hall Inc. USA. Etches, R.J. 1996. Reproductiom in Poultry. UK University Press, Cambridge. Pp. 208 – 232. Gerzilov, V. 2010. Influence of Various Cryoprotectant on the Sperm Motility of Muscovy Semen Before and After Cryopreservation. J. Agri. Sci. and Tech, vol 2 (2) Pp :57 -60.

  Makhafola, M. B., K. C. Lehloenya, M. L. Mphaphathi, A. Dinnyes, and T. L. Nedambale.

  2009. The Effect of Breed on The Survivability and Motility Rate of Cryopreserved Cock Semen. J. Anim. Sci. 39 : 242 - 245. Saleh, D.M., dan Sugiyatno. 2007. Pengaruh Aras Glycerol terhadap Motilitas dan Fertilitas

  Spermatozoa Ayam Kampung yang Dibekukan dengan Nitrogen Cair. Animal Production pp: 45 - 48. Setioko, A.R., P. Situmorang, E. Triwulanningsih, T. Sugiarti, dan D.A. Kusumaningrum.

  2002. Pengaruh Krioprotektan dan Waktu Ekuilibrasi Terhadap Kualitas dan Fertilitas Spermatozoa Itik dan Entog. JITV 7 (4) : 237-243. Seigneurin, F., and E. Blesbois. 1995. Effects of The Freezing Rate on Viability and Fertility of Frozen-Thawed Fowl Spermatozoa. Theriogenology 43 : 1351 – 1358. Tri-Yuwanta, Kustono, C.H. Wibowo. 1998. Pengaruh Pencucian Sel Sperma dan

  Penyimpanan Sperma Ayam Kampung Terhadap Fertilitas. Bulletin Peternakan volume 22 : 64 – 72.