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

  P A A A A A A P

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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

  th Proceedings of the 16 AAAP Animal Science Congress Vol. II

  10-14 November 2014, Gadjah Mada University, Yogyakarta, Indonesia

CONTENTS

ORAL PRESENTATION

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  th Proceedings of the 16 AAAP Animal Science Congress Vol. II

  3URFHHGLQJV RI WKH WK $$$3 $QLPDO 6FLHQFH &RQJUHVV 9RO ,, 10-14 November 2014, Gadjah Mada University, Yogyakarta, Indonesia

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The Effect of Centrifugation Time on the Quality of Domestic Chicken

Spermatozoa Maintained at 5°C

  

Yosephine Laura, Tri Yuwanta and Ismaya

  Faculty of Animal Science, Gadjah Mada University, Yogyakarta, Indonesia Corresponding email: yosephine.laura@ymail.com

  

ABSTRACT

  The objective of this study was to determine the effect of centrifugation time on the quality of sperm diluted in diluter containing physiological sodium chloride and 20% yolk maintained in 5ºC. Sperm was collected from 5 Pelung cocks aged of one year old. The quality of fresh semen was checked. Sperm was centrifugated as well as control (P0), centrifugated 15 (P1), 20 (P2), and 25 (P3) minutes. All sperm treatments were diluted (physiological sodium chloride added by 20% egg yolk) and maintained at 5º C. The quality of sperm such as pH, motility, viability, and abnormality were checked on storage after 0, 3, 6, 9, 12 and 24 hours. All of control and treatments combination were replicated for five replications. The data were analyzed using split-plot design with SPSS 17 for windows. The results showed that time of centrifugation had no effect on sperm quality but the time of storage had a significant effect (P≤0.01) on sperm quality. Time of centrifugation was significantly different (P≤0.05) at 3 hours storaged motility. The averages of pH in each treatment (P0, P1, P2, and P3) were 6.31±0.33; 6.20±0.35; 6.18±0.35; and 6.20±0.35, respectively. The averages of motility in each treatment were 60±14.7%; 65.8±17.1%; 65±20.7%; and 67.2±19.7%, respectively. The averages of viability in each treatment were 54.5±22.3%; 64.1±21.6%; 58.6±22.8%; and 57.4±21.0%, respectively. The averages of abnormality in each treatment were 32.9±18.4%; 36.3±17.6%; 37.4±19.1%; and 36.4±15.3%, respectively. Motility in each treatment (P0, P1, P2, and P3) 3 hours storaged were 69.00±8.94%; 79.00±6.51%; 78.00±4.47%; and 82.00±5.70%, respectively. It can be concluded that centrifugation unable to maintain the sperm quality. The sperm quality decreased gradually during storage.

  Key Words: Domestic chicken, Centrifugation, Storage, Sperm

  

INTRODUCTION

  One of the characteristics of poultry semen is low semen volume with a very high concentration of spermatozoa. It was shown that a little seminal plasma components must supply nutrients to meet the metabolic needs of spermatozoa. Competition between spermatozoa in the use of nutrient sources in seminal plasma can cause deposits metabolic waste and disrupt of other spermatozoa metabolism. Consequently, spermatozoa poisoned by metabolic waste and nutrient deficiencies cause abnormalities even death. Seminal plasma is an important component, but its existence is very limited and deposits of metabolic waste need to be addressed. Centrifugation is a method used to separate the seminal plasma and spermatozoa. Centrifugation causes spermatozoa to settle in the base layer while in the top is seminal plasma. Centrifugation is a mechanical treatment that can cause membrane damage in spermatozoa. The longer the time of centrifugation will cause friction for longer and higher damage. Further examination of the length of time of centrifugation is necessary to be able perfectly separate the seminal plasma and spermatozoa while maintaining spermatozoa quality.

MATERIALS AND METHODS

  The experiment was carried out in the Laboratoroium of Physiologi and Reproduction Animal. Faculty of Animal Science, Gadjah Mada University, Yogyakarta.

  Sustainable Livestock Production in the Perspective of

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  Five roosters (twelve months old) of domestic chicken were used as the semen donors were kept in individual cages. Roosters were provided with commercial feed for reproductive chicken and water ad libitum. Semen was collected and pooled twice a week by the dorso- abdominal massage method.

  Centrifugation method and preparation of diluent

  Medium used for centrifugation was physiological saline diluted 1:9. Treatments were divided into four tubes. The first tube was control tube without washing (P0), the second tube by centrifugation for 15 min (P1), a third tube by centrifugation for 20 min (P2), and the fourth tube by centrifugation for 25 min (P3). Centrifugation was at 1500 rpm with centrifugation machine MLW T52.1. Seminal plasma was removed with micropipette and the diluents were added. Control tube contained 1:9 sperm and diluent. The diluent was made from 80% physiological saline solution was added with antibiotic gentamicin

  5μg/ml and 20% egg yolk of laying hens.

  Sperm examination

  Quality of fresh semen was checked including macroscopic observation of the volume, color, pH, and consistency; then microscopic observations include concentration, motility, viability, and abnormalities. Assessment of sperm quality after treatment were pH, motility, viability, and abnormality during storage at 5° C in 0, 3, 6, 9, and 24 h observations. All microscopic assessments were checked visually with Nikon 120 Microscope.

  Statistical analysis

  All data were counted from 5 replications. The effect of centrifugation time and preservation was analyzed with Split-plot design and Duncan’s multiple range tests (SPSS for windows 17).

  

RESULTS

  Quality of sperm after centrifugation and preservation

  

Table 1. Characteristics of sperm pH after centrifugation and observation in preservation 0, 3, 6, 9,

and 24 hours at 5ºC Observation (h)

  Treatment

  3

  

6

  9

  24 Mean±SD P0 6.44±0.39 6.32±0.37 6.30±0.34 6.26±0.33 6.24±0.32 6.31±0.33 P1 6.38±0.38 6.24±0.39 6.16±0.34 6.12±0.40 6.10±0.34 6.20±0.35 P2 6.30±0.41 6.22±0.44 6.18±0.37 6.10±0.30 6.10±0.33 6.18±0.35 P3 6.34±0.37 6.28±0.39 6.20±0.34 6.12±0.29 6.08±0.34 6.20±0.35

  

Table 2. Characteristics of sperm motility after centrifugation and observation in preservation 0, 3, 6,

9, and 24 hours at 5ºC Observation (h)

  Treatment _{ns r aqr qr q p}

  3

  6

  9

  24 Mean±SD P0 73±9.74 69±8.94 62±4.47 58±4.47 38±13.03 60±14.79 _{r br qr q p} P1 79±8.21 79±6.51 70±10.00 61±11.40 32±15.24 66±17.18 _{r bqr qr q p} P2 83±2.73 78±4.47 68±12.54 63±13.96 33±16.80 65±20.71 _{r br qr q p} P3 84±4.18 82±5.70 68±16.43 62±15.24 41±17.66 67±19.79

  th Proceedings of the 16 AAAP Animal Science Congress Vol. II

  3URFHHGLQJV RI WKH WK $$$3 $QLPDO 6FLHQFH &RQJUHVV 9RO ,, 10-14 November 2014, Gadjah Mada University, Yogyakarta, Indonesia

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Table 3. Characteristics of sperm viability after centrifugation and observation in preservation 0, 3, 6,

  9, and 24 hours at 5ºC Observation (h) Treatment ^ns _{r qr pqr pq p}

  3

  

6

  9

  24 Mean±SD

P0 80.7±11.73 61.7±20.79 55.3±14.53 43.5±16.14 31.3±14.12 54.5±22.32

_{q q q q p}

P1 81.8±6.08 68.6±7.22 71.8±10.35 70.1±12.54 28.5±18.41 64.1±21.65

_{q q q q p}

P2 76.1±10.52 70.1±5.27 63.3±13.53 60.8±23.14 22.8±10.22 58.6±22.89

_{q q q p p}

P3 75.8±12.72 71.1±7.62 65.3±12.99 44.7±14.45 28.6±8.89 57.4±21.09

Table 4. Characteristics of sperm abnormality after centrifugation and observation in preservation 0,

  3, 6, 9, and 24 hours at 5ºC Observation (h) _ns Treatment _{p p p p q}

  3

  

6

  9

  24 Mean±SD

P0 19.7±3.21 25.8±6.39 22.8±5.74 30.8±7.68 64.8±14.79 32.8±18.47

_{p pq pq q r}

P1 16.3±4.41 33.3±10.25 31.4±9.75 38.2±8.17 62.6±13.33 36.4±17.67

_{p p p p q}

P2 24.1±11.11 31.3±12.26 30.3±16.00 35.3±11.38 66.4±12.52 37.5±19.16

_{p p p p q}

P3 26.2±8.70 25.5±4.79 29.5±7.49 39.4±4.77 61.5±9.83 36.4±15.33

Values within each row with different superscripts differ significantly (p,q,r P≤0.05) Values within each column with different superscripts differ significantly (a,b P≤0.01) ns (non-significant)

  

DISCUSSION

  The results showed that sperm centrifugation did not affect the pH of sperm. Centrifugation treatment can not maintain the pH of the sperm. The results differ from Tri-Yuwanta et al. (1998), that the centrifuge sperm cells will slow down decrease in pH value because metabolic processes of sperm cells that produce lactic acid can be suppressed. The decrease in pH affect sperm survival especially during storage. PH values higher or lower than normal sperm causes death sperm (Sujoko et al., 2009). There was a decrease of pH during storage of 24 h. The lowest pH showed in P2 resulted by the production of lactic acid. The results supported by the opinion of Christensen (1995), the low pH of the solution becomes less active sperm, thus reduced by the production of lactic acid. Chicken spermatozoa will produce carbon dioxide and lactic acid that make the lower pH during storage (Thurston, 1995). Centrifugation was not significantly affect the sperm motility. Centrifugation treatment significantly (P ≤ 0.05) affect motility during storage at 3 hours. Treatment of sperm with longer centrifugation time of 25 minutes (P3) tend to produce the most good motility after dilution 3 hours. Centrifugation of sperm could be expected to reduce or even eliminate toxic substances from the seminal plasma and contaminate the remaining sperm cells themselves (Tri-Yuwanta et al., 1998). Components such as spermiophage in the seminal plasma or spermatozoa components as active seminal proteases or fat peroxide can harm sperm during storage (Donoghue and Wishart, 2000). Centrifugation 15 min (P2) and 20 min (P3) can separate the seminal plasma. Storage 24 hours caused a decrease in motility. Storage for 24 hours resulted in the lowest. Accumulation of metabolic waste caused lower motility Christensen (1995) sperm produces metabolic waste that can lower the pH make spermatozoa become inactive and decrease the motility.

  Longer centrifugation did not significantly affect viability. Centrifugation increased the viability but longer can reduce viability. It is supported by Tri-Yuwanta (1998), that the centrifuging sperm is capable of removing toxic substances; whereas seminal plasma were toxic when used in the storage. Control treatment had the lowest viability during storage, it can be affected by toxic substances in seminal plasma that are so toxic to spermatozoa during storage. Centrifugation for 15 minutes tend to be the most excellent in maintaining sperm

  Sustainable Livestock Production in the Perspective of

  6XVWDLQDEOH /LYHVWRFN 3URGXFWLRQ LQ WKH 3HUVSHFWLYH RI Food Security, Policy, Genetic Resources and Climate Change

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  viability. Sujoko et al. (2009), stated that spermatozoa viability can be maintained only at the speed at 400g (1500 rpm) for 15 minutes; prolonged centrifugation can cause friction between spermatozoa with centrifuge tube wall, and medium. It damages to sperm membrane and decrease viability of spermatozoa.

  The results showed that the centrifugation treatment did not affect sperm abnormalities during storage. Centrifugation treatment tends to increase the abnormalities. This is in accordance with the opinion of Sujoko et al. (2009), that centrifugation can decrease sperm binding and sperm membrane lipids due to high frictional forces that disrupted sperm membrane permeability. Sperm covered with a membrane called plasmolema (Hafez. 1987). Plasmolema is the outermost membrane of cells composed of lipoproteins, was selectively permeable, and serves as a transport between cells (Isdarmadi, 2008). The role of the sperm cell membrane is very important, if the cell membrane is damaged then the transport between cells is disrupted and eventually spermatozoa metabolism is also disturbed. Causes impaired metabolism abnormalities in sperm shape.

  

CONCLUSION

  It can be concluded that centrifugation unable to maintain the sperm quality. The sperm quality decreased gradually during storage.

  

REFERENCES

  Christensen, L. V. 1995. Diluents, Dilution, and Storage Poultry Semen for Six Hours. In: Proceedings First International Symposium on the Artificial Insemination of Poultry (editors M.R. Bakst and G. J Wishart) Poultry science Association, Inc. printed in the United States of America.

  Donoghue, A.M., G.J. Wishart. 2000. Storage of Poultry Semen. Animal Reproduction Science 62. Elsevier. United Kingdom. Hafez, E. S. E. 1987. Reproduction in Farm Animals. Lea and Febiger. Philadelphia. Isdarmadi. 2009. Anatomy, Physiology, and Reproductive Cells. Taken from: Accession date 12 April 2011. Iskandar, S.; A.R. Setioko; S. Sopiyana; T. Sartika; Y. Saepudin; E. Wahyu; R. Hernawati dan E. Mardiah. 2005. In situ Conservation of Domestic Chicken (ayam pelung, sentul, dan kedu). Report of Research Activity Ciawi. Bogor. Ismaya; Kustono; S. Bintara; dan D. T., Widayati. 2008. Reproductive Animal Technology.

  Faculty of Animal Husbandry. Gadjah Mada University. Yogyakarta. Sujoko, H; M. Agus Setiadi; A. Boediono. 2009. Sheep Sperm Selection by Percoll Density Gradient Centrifugation Method. J. Veteriner 10:125-132.

  Thurston, R. J. 1995. Storage of poultry semen above freezing for twenty-four to four-eight hours. Pages 107-122 in: Proceedings First International Symposium of the Artificial Insemination of Poultry. M. R. Bakst and G. J. Wishart, ed. Poultry Science Assosiation, Savoy, IL. Tri-Yuwanta. 1998. Effect of Washing Sperm Cells and Sperm Storage on Fertility of Native Chicken . Buletin Peternakan 22:64-72.