BHUTAN JOURNAL of
ANIMAL SCIENCE
Volume Issue March
Department of Livestock
International Standard Serial Number (ISSN) 2520-0194
Key title : Bhutan Journal of Animal Science Abbreviated key title : Bhutan J. Anim. Sci. Parallel title : BJAS
Ministry of Agriculture and Forests Royal Government of Bhutan
All rights reserved.
Department of Livestock, Ministry of Agriculture and Forests, Royal Government of Bhutan, Post Box 113, Postal Code 11001, Tashichhodzong, Thimphu, Bhutan
Dr. Kesang Wangchuk
Dr. Kesang Wangchuk and Mr. Namgay Dorji
Dr. Min Prasad Timsina
Tsang Pa Dung Dok Lhek Dhen Zay Cha Printing Press, Kawajangsa, Thimphu, Bhutan
BJAS is registered permanently with the ISSN International Centre, 45 rue de Turbigo, 75003 Paris, FRANCE
The Bhutan Journal of Animal Science (BJAS), founded in 2017, is an open access English-language journal on animal science. All papers are peer and double blind reviewed. BJAS is supervised by the Department of Livestock, Ministry of Agriculture and Forests, and financed by the Royal Government of Bhutan. BJAS is published annually. It publishes original work that makes a significant contribution to understanding and enhancing the animal production practices in smallholder systems in the mountains. The original works encompass the social, cultural, economic, and policy influences affecting the use and management of plant and animal resources. BJAS publishes research, reviews, reports on all aspects of ruminants, piggery, poultry, aquaculture, and apiculture. BJAS also accepts livestock associated research reports on climate change, sustainable development, rural livelihood, socio-economics, and policy. BJAS requires academic papers and research reports to display universal, strategic and innovative characteristics in both theory and practice. BJAS is a forum for sharing information between researchers, policy makers and practitioners.
Articles in BJAS present the results of original research, contributions to theory or new conclusions reached from the review of a topic. Their structure conforms to that of standard scientific articles and writing style is made clear and concise. Results are critically analyzed and objectively presented.
Major topics published • Animal production • Animal genetics and biotechnology • Forage and animal nutrition • Post-production and management • Socio-economics • Policy • Animal health
Bibliographic Details ISSN: 2520-0194; Frequency: Annual; Volume: 2; Issue: 1
Editorial Board
Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, 2018
Chief Advisor DR. TASHI SAMDUP
Editor-in-Chief DR. KESANG WANGCHUK
Associate Editors
Editorial correspondence should be addressed to: The Editor-in-Chief, Bhutan Journal of Animal Science, Department of Livestock, Ministry of Agriculture and Forests, Thimphu, Bhutan.
Vol. 2 No.1 March 2018 Bhutan Journal of Animal Science (BJAS)
ANIMAL GENETICS AND BIOTECHNOLOGY 1-6 1. Decades of artificial insemination: bittersweet experiences on cattle breeding in Bhutan LHAM TSHERING AND NAR B TAMANG 2. Semen characteristics of three strains of Bhutanese indigenous chicken 7-13 SANGAY DORJI AND NEDUP DORJI 3. Seminal attributes, cooling procedure and post thaw quality of semen of 14-19 indigenous Khari buck of Nepal PANKAJ K JHA, SAROJ SAPKOTA, DIL B GURUNG, RAJU KADEL, NEENA A GORKHALI AND BHOLA S SHRESTHA
ANIMAL PRODUCTION 4. Length-weight relationship and relative condition factor of Golden Mahseer 20-25 (Tor putitora Hamilton 1822) under pond-reared condition in southern foothills of Bhutan NAMGAY DORJI, DRUKPOLA, JAMYANG NORBU AND NETEN 26-30 5. Effects of stocking density on performance of cultivable carps in Southern Bhutan PEMA THINLEY, DRUKPOLA AND NAMGAY DORJI 31-36 6. Exotic pigs receive different fattening durations from Bhutanese farmers TENZIN PENJOR, GYEMBO TSHETEN, PEMA SHERAB AND MIN P TIMSINA 37-41 7. Production performances of crossbred pigs in government farms LOKEY THAPA 42-48 8. Effects of weaning age on growth, health and mortality of piglets in government pig farm SINGAY OM, PEMA SHERUB, TSHERING GYELTSHEN AND MIN P TIMSINA 49-52 9. Breed preference and breeding practices of dairy farmers in Bhutan DEKI CHODEN AND NAR B TAMANG 53-56 10. Effects of poultry feed and stocking density on broiler performance TASHI JAMTSHO AND SURYA BC RAI 57-61 11. High fiber diet as molt inducers for commercial layers under deep litter system in Bhutan TASHI JAMTSHO AND NIDUP DORJI
FORAGE AND ANIMAL NUTRITION 62-65 12. Effects of Total Mixed Ration (TMR) differ between progressive and less progressive dairy farms WANGCHUK AND JAMBAY GYELTSHEN 66-69 13. Herbage yield and nutrient content of Oat var. Stampede under different irrigation intervals DORJI WANGMO AND REKHA CHHETRI 70-74 14. Dry matter content of fodder resources used by dairy farmers’ group in West- Central Bhutan AITA K BHUJEL, UGYEN NAMGYAL AND BIR D RAI
ANIMAL HEALTH 75-81 15. Microbiological quality of raw milk in Bhutan SURYA BC RAI, PUSPA M SHARMA, TENZINLA, PUNYA MATA, PEMA TSHOMO AND BHAKTA B GURUNG 82-86 16. Foot and Mouth Disease (FMD): outbreak containment at cattle herd level in Samdrup Jongkhar district LUNGTEN LUNGTEN, TENZIN TENZIN, KAROON CHANACHAI AND TSHEWANG RABGAY 87-91 17. Plant extracts as alternatives to anti-anthelmintic drugs: findings from Maedwang subdistrict, Thimphu NIDUP DORJI AND PHUB DORJI 92-99 18. Bursal disease outbreak in vaccinated young poultry flocks in southern districts of Bhutan RATNA B GURUNG, SONAM TENZIN, RINZIN PEM, NAR K THAPA, TENZIN GEMBO AND KINZANG DUKPA 100-106 19. Leptospirosis associated abortion in cows of National Jersey Breeding Centre, Samtse RATNA B GURUNG, TENZINLA, DENDUP TSHERING, SANGAY TENZIN, LUNGTEN, DAL B CHHETRI, SONAM WANGCHUK AND KINZANG DUKPA
POST-PRODUCTION, SOCIO-ECONOMICS AND POLICY 107-111 20. Dairy farming enhances household income in peri-urban and rural areas of Choekor in Bumthang, Bhutan GYEM TSHERING
SHORT COMMUNICATION AND REPORT 112-114 21. Microbial load in local and imported raw milk TENZIN PENJOR AND TSHERING GYELTSHEN 115-116 22. Surgical removal of teat siphon from cow’s udder TENZIN WANGCHUK AND PREM B GIRI 117-119 23. Effects of body condition score on postpartum estrus cycle in dairy cows PEMA UGYEN, PENJOR AND RINZIN DORJI 120-122 24. Moisture content of local cottage cheese and butter in Western Bhutan SONAM YANGCHEN, PHUNTSHO T NORBU AND Y NAKANISH
REVIEW PAPER 25. Anti-Microbial Resistance (AMR) in Bhutanese livestock: a review 123-126 NIRMAL K THAPA, PUSPA M SHARMA, NARAPATI DAHAL, SURYA B CHAMLING, VIJAY RAIKA, JAMBAY DORJEE, RATNA B GURUNG AND KINZANG DUKPA
INSTRUCTIONS AND GUIDELINES FOR SUBMISSION OF ARTICLES 127-129
Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 1-6, 2018
Full length paper decades of artificial insemination: bittersweet experiences on cattle breeding in Bhutan
LHAM TSHERING* AND NAR B TAMANG
National Dairy Research Center, Department of Livestock, Ministry of Agriculture and Forests, Yusipang, Thimphu, Bhutan
*Author for corespondence: [email protected]
Copyright © 2018 Lham Tshering. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: The main objectives of the study were to determine the number of inseminations performed and progenies born between 1987 and 2016, compare Artificial Insemination (AI) performance in different regions, among districts and AI Outreach Stations (AIOSs), and estimate contribution of AI program to total milk production and national economy. Data was collected in 2016, covering 20 districts and 106 AIOS. Establishment of 106 AI Out Reach Centre in 20 districts provided uninterrupted AI services, enabling AI performed to exceed 160,070, resulting in birth of 50,808 improved calves since 1987. Western region had the highest number of progenies born (35%). East Central Region had the lowest with 15% of total calves born in the region. Among 20 districts, Paro was the best performer with 16,360 inseminations done with 5,829 progenies born. Gasa district performed poorly with 24 progenies born out of 73 inseminations performed. The average number of inseminations per month and corresponding progenies born was highest for AIOS located at Veterinary Hospital (VH) in Paro, followed by Veteninary Hospital (VH) in Tsirang, Extension Centre (EC) in Chaskhar, VH in Thimphu and EC in Deothang. VH in Gasa, EC in Tashiding, EC in Bjemina, EC in Goshi had less than one insemination per month. At the national level, the average number of AI performed was about seven times per month but only 50% of the AIOS exceeded this minimum recommended number of AI to be performed per AIOS. The national average success rate was 37% and the corresponding total progenies born should have been 56,910. However, due to lack of proper recording, the number of progenies born stands underestimated by 14.2%. The study concluded that, for higher output of National AI Program, there is a need to improve progeny recording system, improve skills of AI Technicians, train community AI Technicianss, relocate or close under-performing AIOS and take joint ownerships by all stakeholders at national, regional and district level.
Keywords: Artificial Insemination; breed improvement; economy; progeny.
1. INTRODUCTION as a primary breeding tool for genetic upgradation of cattle (Wardena et al. 1998). In Bhutan, the AI Artificial Insemination (AI) is the most efficient program has been implemented in a planned manner reproductive technology accepted by the scientific since 1987. In areas where AI is not feasible, quality community for breed improvement and breeding bulls were supplied from government cattle enhancement of dairy productivity. AI is considered farms and through contract heifer and bull 1
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 1-6, 2018 production programme to disseminate superior Buffaloes was introduced for the first time in the genetic trait of selected elite males for breed country. improvement (Tshering 2017). The number of With increasing urban demand for dairy inseminations and progenies recorded has increased products in the country, AI technology played an over the years. Although the improvement has not important role in gradual transition from subsistence been very drastic, the trend of steady increase is an to a market-oriented production system over the indication of success, taking into account the years. However, a comprehensive review of convergent factors called the chain of fertility. The National AI Program and its impact on dairy chain of fertility includes the sexual health of the development and national economy has not been bull, frozen semen quality, expertise of AI undertaken. Therefore, a study was conducted to Technician and the ability of the farmers to detect review AI program since its inception. The study the animal in heat and take the animal at the right objectives were to evaluate the number of time for insemination. Nevertheless, since the inseminations done and progenies born between commencement of AI programme, there has been 1987 and 2016, compare AI performances in steady increase in AI coverage. It was due to different regions, districts and AIOS, and estimate innovative approaches such as initiation of Contract contribution of AI program to total milk production Heifer, Bull Production Program in 2000 and use of and national economy. progeny tested semen for this program, which triggered formation of 194 Dairy Farmers’ Groups 2. MATERIALS AND METHODS and five Dairy Farmers’ Cooperatives across the 2.1 Data collection and analysis country (NDRC 2016). The locations of AIOSs are presented in Figure 1. To take AI services closer to farmers’ door- Data collection was carried out in 2016, covering 20 steps, over 82 Community AI Technicians were districts and all 106 AIOS. The study team visited all trained and deployed since 2010 to complement and AIOS. AI done and progeny records maintained at supplement AI Technicians (Tshering et al. 2016). the AIOS from 1987 to 2016 were reviewed. The Since 2015, to meet the changing needs of farming individual records were evaluated. Informal communities, sex sorted semen was tried in village discussions were held with all AI technicians to herds to ensure higher number of female born understand the constraints faced. Impressions and (Tshering 2015). To harness the opportunity existing observations from the field were described. Data in science and technology, Embryo Transfer obtained from the records were entered in Microsoft Technology was vigerously pursued for faster Excel sheet and analysed using MS Excel and SPSS. multiplication of elite germplasm since 2015. By The percentage of AI done and progeny born in each 2017, the Progeny Testing Scheme was initiated to develop resilient cattle breed for Bhutan and AI in
Figure 1: Artificial Insemination Outreach Stations(AIOSs) in the country as of June 2017 (Black dots represent AIOSs).
Tshering and Tamang (2018) 2
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 1-6, 2018 regions of the country were calculated. Descriptive advantage of the technology for upgradation of local and inferential statistics were used. cattle population in the region.
3. RESULTS AND DISCUSSION 3.3 AI done and calves born in Districts Figure 4 presents the details of AI and projenies born 3.1 Comparison of AI technology application in in 20 districts. Records from 1987 to 2016 showed four regions of the country that, across 20 districts of the country, Paro was the Comparison of AI technology application in four best performer with 16,360 inseminations done with regions of the country revealed that western region 5,829 progenies born. Gasa performed poorly with was ahead in adopting the AI technology with 24 progenies born out of 73 inseminations done. The highest number of progenies recorded (17,725) remoteness of Gasa district, coupled with cold (Figure 2). East-central region lagged behind with weather could have had adverse effects on 7,739 progenies born. This could be because western technology adoption. region is more accessible with motorable road; Average insemination per month and farmers are innovative and forthcoming to embrace corresponding progenies born were highest for the the technology in view of better market for fluid AI station located at the Veterinary Hospital (VH) in liquid milk. The success rate of AI is reported to vary Paro. The lowest AI per month and projenies born from province to districts depending on AI coverage were observed for VH in Gasa, Extension Centre (Wardena et al. 1998). (EC) in Tashiding, EC in Bjemina, EC in Goshi,
180000
Total no.of AI done Total progeny recorded 160070 160000
140000
120000
100000
80000
60000 49679 50629 50808
40000 30272 29490 17725 20000 15783 9561 7739
0 Western West Central East Central Eastern Region Overall Region Region
Figure 2: Region-wise comparison of AI technology uptake (1987-2016).
where average insemination per month is less than 3.2 Regional share of progenies born through AI one. The share of progenies born out of AI in four regions Paro, Bumthang, Mongar, Punakha, Tashigang, is presented in Figure 3. Western region had the Thimphu, Samtse and Samdrup Jongkhar districts highest share of progenies born (35%) through performed extremely well whereas Gasa, Lhuentse, application of AI technology, followed by eastern Dagana, Sarpang, Haa were far below average. AI region (31%). East-central region had lower performances of AIOSs that are not upto the mark adoption of technology, resulting in only 15% of need close monitoring by respective Regional total calves born in the country. Hence, east-central Livestock Development Centres and appropriate region needs to provide extra efforts to take
Tshering and Tamang (2018) 3
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 1-6, 2018 remedial measures must be taken to accelerate the 3.4 Trend in application of AI technology technology uptake. The yearwise linear trendline of adoption of technology showed an upward trend indicating that technology application by farmers increased steadily over the years (Figure 5). Technology usage picked up well and peaked in 2003-2004 with about 6000 inseminations done but failed to rise further in the Western Eastern subsequent years and remained more or less region region constant. This could be because the push to 35% 31% popularize the technology, facilities and support for the program could have remained static and the awareness level amongst famers could also have remained the same.
West East central 3.5 Trend in adoption of AI Technology in four central regions of the country region region 15% The assessment of AI technology adoption across 19% four regions of the country revealed that west central
region, after a fall in 1999-2000, saw a steady rise in Figure 3: Share of progenies born out of AI in AI technology adoption. In east central region, the four regions. AI technology adoption declined since 1998, which could be due to gradual withdrawal of donor fund support to AI program. Hence, the financial support
18000 No.of AI done Progenies recorded 16360 16000 13389 14000 13095 12000 10360 9759 9139 9691 10000 8134 8000 6192 6000 4120 3527 3263 4000 2267 1920 1592 1491 1350 2000 828 73
0
Haa
Paro
Gasa
Samtse
Dagana Tsirang
Chukha
Trongsa Sarpang
Mongar
Punakha
Lhuentse
Thimphu
Wangdue
Bumthang
Zhemgang
Trashigang Tasiyangtse
Pema Gatshel Pema
Figure 4: Districtwise comparison of inseminations done and projenies born.
7000 Total AI done Total Progeny born Linear (Total Progeny born ) 6000 5000 4000 3000 2000
AI & Progeny born Progeny & AI 1000 0
2002-03 1987-88 1988-89 1989-90 1990-91 1991-92 1992-93 1993-94 1994-95 1995-96 1996-97 1997-98 1998-99 1999-00 2000-01 2001-02 2003-04 2004-05 2005-06 2006-07 2007-08 2008-09 2009-10 2010-11 2011-12 2012-13 Year Figure 5: Yearwise adoption (1987-2013) of AI technology across the country. Tshering and Tamang (2018) 4
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 1-6, 2018 and incentives for adoption of AI technology need to 2007-2012 was 36%, which steadily improved to be continued to promote its uptake. 38% in 2012- 2016 (Table 1). The success rate from nine years national 3.6 Comparision of AI performace in AI average (2007-2016) was 37%, which is higher than Outreach centres the success rate of 34.3% reported from Sri Lanka The average number of inseminations per month and (Wardena et al. 1998). This is indicative of corresponding progeny born was highest for consistent effort of National Dairy Research Centre Veterinary Hospital (VH) in Paro, followed by VH and its allies to reach AI technology closer to dairy in Tsirang, Extension Centre (EC) in Chaskhar, VH farming communities. Success recorded in Bhutan is in Thimphu and EC in Deothang (Figure 6). On the mostly with one time insemination of cattle in heat contrary, the average inseminations per month was brought to AIOS by farmers. However, studies less than one for VH in Gasa, EC in Tashiding, EC have shown that success rate can be improved by in Bjemina, EC in Goshi, which needs to be about 4% if there are two inseminations in the reviewed with caution. About 50% of the AIOS morning and evening (Graves et al. 1997). Thus, performed below the national average of 7±1 increasing the success rate could be possible if two insemination per month. inseminations are followed after considering cost implications. 3.7 Success rate of AI program at national level Success rate was measured by accounting number of 3.8 Contribution of AI technology to National inseminations done and progenies born. The overall Economy success of 31% was recorded till June 2012 because On average, 8,398 AI was performed and 3,107 the initial take-off of AI program in late 1980s and progenies were born annually. Considering 20-25 1990s was slow. However, the success rate from July years life expectancy of cattle (Banerjee 1991), the
35 Average AI per month 30 25 20 15 10 5
0
No. of inseminations per inseminations of No.
DVH
RNR-EC…
DVH Haa DVH
LEC Taloo LEC
LEC Guma LEC
LEC Gaselo LEC
DVH Tsirang DVH
RNR-EC Buli RNR-EC
LEC Dagapela LEC
LEC Chaskhar LEC
LEC Phangyul LEC
LEC K/Rabten LEC
LEC Yangneer LEC
CRC Wangkha CRC
NAH Thimphu NAH
RNR-EC Goshi RNR-EC
RNR-EC Shaba RNR-EC RNR-EC Dotey RNR-EC Ramtokto DVH
LEC Chasilakha LEC
DVH Bumthang DVH Bomdeling LEC
RNR-EC Kabisa RNR-EC
RNR-EC Samtse RNR-EC
LEC Tangmachu LEC
DVH Trashigang DVH
LEC Thinleygang LEC
RNR-EC Thangbi RNR-EC
RNR-EC Drepong RNR-EC
Brown Swiss farm Swiss Brown
DVH Pemagatshel DVH RNR-EC Yoedtseltse RNR-EC NNBF, Tashiyangphu NNBF, Figure 6: AI performaces of AI Outreach centres in Bhutan.
Table 1: AI success rates in 10th and 11th Five Year Plans. AI progress 2007-2012 AI progress 2012-2016 Region of Bhutan Total AI Progeny Success Total AI Progeny Success done born rate (%) done born rate (%) Western 9258 3530 38 8378 3221 38 West Central 6281 2357 38 7633 3068 40 East central 7219 2032 28 6866 2496 36 Eastern 9681 3851 40 10716 4037 38 Average success rate 36 38 Data source: District AI records, Success rate is progeny born devided by total AI ×100
Tshering and Tamang (2018) 5
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 1-6, 2018 estimated 10,312 females born out of AI were below Graves WM, Dowle HH, Lamar KC, Jhonson DL, 15 years and are still in production, producing Saxton AM, and Montegomery MJ (1997). approximately 22,583 MT milk (6kg cow-1 day-1, Effect of Artificial Insemination once versus (price Nu. 45 kg-1), ploughing back Nu. 1.016 twice per day. Journal of Dairy Science, 80 (11): Billion to dairy farmers annually. Of the annual milk 3068-3071. production of 47,270 MT in 2016 (DoL 2017), AI Tshering L (2017). Evolution of Artificial technology contributed 48% of total milk produced Insemination Program in Bhutan, A in the country. Thus, the positive impact of AI comprehensive review on its historical technology application for cattle breed improvement Prespective, National Dairy Research Centre, and its contribution to national economy are Yusipng, In press. substantial (Figure 7). Tshering L, Rai DB, and Timsina MP (2016). Community AI Technicians: Striving to reach Breeding AI services closer to farmers’s door steps. bull Sonam Drubdey, Ministry of Agriculture and projeny Forests, Thimphu, Bhutan. 14% Tshering L, Rai DB and Dorji (2016). Improving Projeny reproductive efficiency of non clycling cows born through estrous synchronization. Annual Local from AI Report, National Dairy Research Centre, cattle 48% projeny Yusipang. 32% Imported Tshering L (2015). Report on use of sex sorted cattle semen at NJBC Samtse and CRC Wangkha, projeny National Dairy Research Centre, Yusipang. 6% Wardena AH, Alexander PA (2018). Artificial Inseminaion of Cattle in Sri Lanka, Status, Performance and Problems. Available at: Figure 7: Contribution from AI born progeny to http://www.iaea.org/inis/collection/NCLCollect overall milk production. ionStore/_Public/32/032/32032357.pdf. (Accessed on 22 March, 2018). 4 CONCLUSION Prognies born from AI have substantially contributed to milk production and national economy. However, the follow up on progenies born is inadequate in many of the AIOS and not all progenies born are eartagged and recorded. This is underestimating the success rate of the national AI program. To enhance AI performances, districts performing well should be rewarded and trailing districts should be supported. There is a need to strengthen the capacity of staff placed at AIOS. Increased development activities in the subdistricts and multi-tasking of Extension Agents are reported to be the reason for less focus on AI and progeny recording. Joint ownerships of AI and dairy breed improvement programs is a way forward to achieving higher output. Unless such collective efforts are made and backed by required resource, dairy breed improvement will continue to show low impact, compeling farmers to import cattle from outside the country.
REFERENCES Banerjee GC (1991). Textboo of Animal Husbandry, Oxford and PBH publications, New Delhi DoL (2017). Annual Livestock Statistics, Department of Livestock, Thimphu, Bhutan.
Tshering and Tamang (2018) 6
Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 7-13, 2018
Full length paper Semen characteristics of three strains of Bhutanese indigenous chicken
SANGAY DORJI1* AND NEDUP DORJI2
1Dzongkhag Livestock, Department of Livestock, Ministry of Agriculture and Forests, Zhemgang, Bhutan. 2College of Natural Resources, Royal University of Bhutan, Lobesa, Punakha, Bhutan.
*Author for correspondence: [email protected].
Copyright © 2018 Sangay Dorji. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: The objectives of the study were to compare the semen characteristics of Seim, Yubjha Naap and Baylaity chicken and correlate semen characteristics with secondary sexual traits. A total of 12 cocks (4 cocks from each population) were used for semen analyses, which included semen volume, semen pH, semen color, sperm concentration, sperm motility and semen mass activity. Semen was collected by abdominal massage method. There was a significant difference in semen volume, concentration and motility among the chicken populations but there was no significant difference in semen color, mass activity and semen pH. Semen colors were all creamy white. The highest semen volume was recorded in Seim (0.55±0.4 mL) and the lowest in Yubjha Naap (0.37±0.02 mL). Seim also had the highest sperm concentration (6.34±0.49 billion mL-1) while Yubjha Naap had the lowest (4.45±0.37 billion mL-1). Seim had the highest semen motility (75.00±1.22%) while Yubjha Naap (64.25±2.59%) had the lowest motility. The mass activities in Seim, Yubjha Naap and Baylaity were 2.75, 2.5 and 3.75, respectively. All chicken populations had an average pH closer to neutral (Seim-7.12±0.12, Yubjha Naap-7.47±0.09 and Baylaity-7.15±0.06). Semen volume, semen concentration, semen mass activity and semen motility were positively correlated with secondary sexual traits. However, semen pH was negatively correlated with all secondary sexual traits. The strongest correlation (r=0.76) was observed between beak length and semen concentration. The semen of all three strains of indigenous Bhutanese chicken were found suitable for preservation and therefore, can be cryopreserved. Moreover, the secondary sexual traits such as body weight, comb size and beak length can be used to predict semen quality of Bhutanese cocks.
Keywords: Chicken; indigenous cocks; semen characteristics; secondary sexual traits.
1. INTRODUCTION Gyeltshen 2012). Chickens are kept for egg, meat, feather, manure and social rituals. Poultry farmers Chicken (Gallus domesticus) is an important seldom sell or slaughter indigenous chicken in livestock component in Bhutanese farming Bhutan (Dorji and Dorji 2015). Indigenous community (Nidup and Tshering 2007). Poultry chickens are highly adapted to local environment farming is considered as “an entry point for poverty (Brion 2012; FAO 2014). For example, Bhutanese reduction” and “gateway to national food security”, chicken are tolerant to Infectious Bursal Disease particularly in developing countries (Dorji and (Dorji et al. 2016).
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Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 7-13, 2018
Indigenous chickens are reared under semen samples at an interval of three days were scavenging system and are provided night shelter collected from each cock to obtain accurate data with little or no feed supplements. However, the (Tarief et al. 2013). hen lays less number of eggs and reaches marketable age at two to three years (Nidup and 2.2 Management of experimental birds Tshering 2007). Therefore, the government Each cock was kept in individual cage 10 days initiated several development strategies to increase prior to semen collection, in order to prevent poultry production in Bhutan, such as introduction fighting and ensure access to feed and water. The of commercial layers and subsidies for starting battery cage (52 cm × 45 cm × 38 cm) was small-scale poultry farms. These initiatives resulted constructed using locally available materials (Tarif in country achieving 100% self-sufficiency in egg et al. 2013). The hens were also introduced inside and 47% self-sufficiency in chicken meat since the house to induce semen production in cocks. 2012 (Gross National Happiness Commission About 16 hrs light was provided both in adaption [GNHC] 2012). On the contrary, the increasing and experiment period (Bacon et al. 2000). The commercial poultry farming may lead to dilution of birds were fed with complete commercial layer genetic resource base of indigenous chicken. poultry feed as feeding layer feed to roosters does Conservation and sustainable utilization of not affect semen quality in male (Hubbard 2011). biological diversity is crucial for meeting the food, Individual birds were fed twice a day (morning and health and other needs of growing world human evening). Clean drinking water was provided population (FAO 2014). More importantly, the throughout the study period. The cocks were de- climate change has posed threat to extinction of wormed during adaption period. one-fourth of Earth’s species (FAO 2015). Global warming causes heat stress to animals and impairs 2.3 Semen collection production, reproduction, health and immune Feathers around the cloacal region were trimmed as response (Nardone et al. 2010). Climate change per Peters et al. (2008) to prevent sample leads to outbreak of new diseases and parasites contamination. Cocks were trained for 10 days (Verge et al. 2009). Therefore, to sustain livestock prior to semen collection (Tabatabaei 2009). Single farming in the face of changing environment, it is ejaculation through abdominal massage technique important to maintain diverse genetic resources. In (Lake 1960) from each cock was collected at Bhutan, the National Biodiversity Center (NBC) specific time, at an interval of three days. Each has initiated conservation of poultry genetic cock was massaged at the back and stroked close to resources since 2008, through in-situ conservation its tail with slight finger pressure around the base of and gene cryopreservation (Tamang 2011). To the tail. The abdomen of the cock beneath the vent conserve good semen quality in the cryobank, it is was pressed with thumb and it caused immediate important to assess the semen characteristics of release of semen from ducts deferens. The semen Bhutanese indigenous chickens. Therefore, this was then gently squeezed into the conical graduated study was conducted with the objective to examine collecting tube. The excess squeezing was avoided the semen quality and quantity of three strains of to prevent bleeding (Lake 1962 as cited by Peters et indigenous Bhutanese chicken. al. 2008).
2.4 Semen evaluation 2. MATERIALS AND METHODS The semen evaluation was conducted at room 2.1 Study area, population and sample size temperature. The Standard Operating Procedure The study was conducted at NBC, Serbithang, and laboratory protocol of NBC were followed Thimphu. The study area is located at 27025’12.2” throughout the study period. Using a conical N, 0890 39’04.6” E. It is located at an altitude of graduated poultry semen collecting tube in millilitre 2,487 meters above sea level with an average (mL), the per ejaculation volume of semen from annual temperature of 11.8°C. It has an average each cock was measured fresh, when aspirated from annual relative humidity of 73.9% (NBC 2011). cloacal vent. For semen color, the visual inspection The sample cocks were sourced from Dagana of fresh semen was conducted within three minutes and Tsirang districts as they have maximum of milking. The color of semen was scored as numbers of indigenous chicken (DoL 2014). A total 1=Creamy white, 2=Between creamy white and of 12 cocks (4 native black [Yubjha Naap], 4 native opaque and 3=Opaque (Peters et al. 2008). The red [Seim] and 4 short legged [Baylaity]) were used semen concentration was determined by AccuRead for assessing sperm characteristics. The selected photometer (IMV Germany), which was maintained experimental cocks were sexually matured, healthy at 37°C. About 2 mL of 0.9% normal saline (NS) and free from physical injuries. Five replicates of was pipetted into the 15 mL cuvette. The cuvette was then placed in AccuRead photometer and was Dorji and Dorji (2018) 8
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 7-13, 2018 calibrated to zero. About 10 µL of fresh semen was 2.6 Data Analysis added into the cuvette with normal saline. The Data collected was entered in Microsoft Excel sheet cuvette was taken out from AccuRead photometer, and analyzed with SPSS version 21. Analyses of covered with aluminum foil and then shaken gently variance (ANOVA) was used to test differences in until the semen was mixed thoroughly with normal parameters among strains. Differences were saline. The cuvette with semen and normal saline considered significant when p value was less than mixture was placed into AccuRead photometer and 5%. Pearson’s correlation test was conducted to pressed “Measure” and recorded semen establish correlation between semen characteristics concentration in billion per mL. A drop of semen and secondary sexual traits. was placed on a clean dry preheated slide at 37°C. The semen was covered with a warm glass cover 3. RESULTS AND DISCUSSION slip to ensure uniform spread of semen and prevent 3.1 Semen color from quick drying. A microscope magnification of All semen colors were creamy white, which was – 40X was used to observe the semen motility, also reported in Nigerian frizzle, naked neck and which was expressed as percentage of mobile Nera black chicken (Peters et al. 2008) and RIR spermatozoa with moderate to rapid progressive rooster in India (Churchil et al. 2014). The movement (Tarif et al. 2013). With the same slide, similarity could have occurred due to similar semen mass activity was scored in the scale: feeding management as the semen color is 1=number of perceptible motion, 2=few dependent on feed (Dhawale 2012). spermatozoa moving without forming any waves,
3=small with slow moving waves, 4=vigorous 3.2 Semen volume movement with moderately rapid wave and eddies There was no significant difference in semen and 5=dense, rapidly moving waves and eddies volume between the populations except for Seim (Tarif et al. 2013). and Baylaity (Figure 1). Seim produced the highest The pH of semen was determined by using semen volume of 0.55±0.40 mL, followed by indicator paper strips (INDIKROM PAPERS). Baylaity with 0.45±0.03 mL, and Yubjha Naap with About 3-4 drops of fresh semen was added on the 0.37±0.02 mL. The semen volume of all three indicator paper strips using micro pipette. The populations falls within the range of 0.37±0.02 to semen was gently pressed with the help of glass 0.73±0.01 mL as found for Nigerian local chicken slide to obtain uniform spread of semen over the (Peters et al. 2008). The semen volume of indicator paper strip. The paper strip was then air- Bhutanese chicken was more than the Malaysian dried for 1-2 minutes. The visual observation and domestic chicken (0.33±0.16 mL), Bantham the reading were recorded by comparing the color chicken (0.29±0.18 mL) and Red Jungle Fowl obtained with the reference color and pH reading given on the indicator paper strip cover (Kamar et al. 1979). The procedure was repeated three times 0.7 with different paper strips in each sample to confirm the reading. 0.6 a
2.5 Secondary sexual traits measurements 0.5 a The secondary sexual traits were measured after b completing milking semen from each cock. The 0.4 traits were measured at the beginning (day 0) and at the end of study (day 12). Electrical weighing
Semen volume (mL) volume Semen 0.3 balance was used to measure the linear body weight of the cocks. Body weight was measured after semen milking and before feeding. Comb size, 0.2 comb length and comb height were measured (Dhawale 2012). The comb height was measured 0.1 from where the comb met the head to the top of the highest spike, while the comb length was measured 0 from back end to front end, using a measuring tape Seim Baylaity Yubjha (Navara et al. 2012). Beak length was measured as Naap the distance between the tip of the beak and the base, using measuring tape (Udeh et al. 2011). Figure 1: Semen volume of Seim, Yubjha Naap and Baylaity (mean±se of mean). Means with different letters differ significantly (p≤0.05).
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(0.10±0.10 mL) (Malik et al. 2013). The higher 3.4 Semen mass activity semen volume in this study could be due to Figure 3 presents the semen mass activity of differences in body weight (Dhawale 2012). The chicken strains. There was no significant difference cocks in Malaysian study were lighter in weight, in semen mass activity among three populations. and semen volume was positively correlated with The mass activities in Seim, Yubjha Naap and body weight. Baylaity were 2.75, 2.50 and 3.75, respectively. 3.3 Semen concentration Semen mass activity was better in Baylaity, There was a significant difference (p≤0.001) in followed by Seim and Yubjha Naap. It is important semen concentration between Seim and Yubjha to have high semen mass activity for fertility due to Naap. Semen concentration also differed long storage of semen in the utero-vaginal junction significantly (p≤0.05) between Seim and Baylaity. of female before it reaches fertilization site However, no significant difference was observed (Tadondjou et al. 2014). The semen mass activities between Yubjha Naap and Baylaity. Highest sperm concentration was produced by Seim population of all populations were close to Indonesian local with a mean concentration of 6.34±0.49 billion mL- chicken of 2.5 and 3 (Almahdi et al. 2014). 1 and least by Yubjha Naap with a mean However, the mass activities of Bhutanese concentration of 4.45±0.37 billion mL-1 (Figure 2). indigenous chickens were lower than the range of Semen concentration of all three populations fell 3.80±0.10 to 4.00±0.00 found in four breeds of -1 within the range 1-7 billion sperm cells mL as cocks in Bangladesh (Tarif et al. 2013). The low reported by Peter et al. (2008) in Nigerian frizzle, temperature in the study area as compared to naked neck and Nera black chicken. Differences in semen concentration among the breeds were also Bangladesh probably contributed to the lower mass observed by Malik et al. (2013) in domestic activity (Gebriel et al. 2009). There was no chicken in Malaysia. However, all cell counts were variation in mass activities among the populations, higher than the Red Jungle Fowl of 4.44×109 cells which indicates that the semen mass activity was -1 9 mL and Malaysian domestic chicken of 1.83×10 not affected by the genetic makeup, because all the cells mL-1 (Malik et al. 2013). On the other hand, feeding and management practices were same. Zhang et al. (1999) reported no effect of strain on semen concentration in broiler breeds. The variation in semen concentration among breeds 4.00 indicates the influences of genetic makeup of the birds (Dhawale 2012). 3.50
8 3.00
7 a 2.50
109) × 6 2.00
b Mass activity score activity Mass 5 b 1.50
4 1.00
3 0.50 Semen concentration ( concentration Semen 2 0.00 Seim Yubjha Naap Baylaity 1 Figure 3: Semen mass activity.
0 3.5 Semen motility Seim Baylaity Yubjha Naap The semen motility among strains is presented in
Figure 4. Semen motility did not differ significantly Figure 2: Semen concentration (×109) among between Seim and Baylaity. However, the semen populations (mean±se of mean). Means with motility of Seim and Baylaity was significantly different letters differ significantly (p≤0.05). higher (p≤0.00) than Yubjha Naap. Bhutanese chicken semen motility is reported to fall within the Dorji and Dorji (2018) 10
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 7-13, 2018 range of 49.40±6.03% - 96.00±2.27% found in local chicken (Peters et al. 2008; Ajayi et al. 2011), Malaysian chicken (Malik et al. 2013) and Bangkok chicken and Arab chickens in Indonesia 62.55±10.55% - 87.35±10.12% found in Nigerian (Almahdi et al. 2014). local chicken (Peters et al. 2008). However, the semen motility of indigenous Bhutanese chicken 3.7 Relationship between semen characteristics was less than that of Bangkok chicken and secondary sexual traits (84.00±2.23%), Lingnan chicken (84.00±2.23%), The relationships among traits and semen Kedu chicken (84.00±2.23%) and Arab chickens characteristics are shown in Table 1. Semen (80.00±7.07%) in Indonesia (Almahdi et al. 2014). volume, semen concentration, semen mass activity The lower semen motility in this study could be due and semen motility were positively correlated with to age of the cocks. The best quality semen is secondary sexual traits. However, semen pH was produced by the cock at an age of 10-20 months negatively correlated with all secondary sexual (Nalbandov 1990). The cocks used in this study traits. The strongest correlation was observed were all older than 30 weeks, whereas the cocks between beak length and semen concentration. used by Almahdi et al. (2014) were 10-20 weeks old, which might have affected the semen motility. 7.6
3.6 Semen pH 7.5 -ns- There was no significant difference in pH among the strains (Figure 5). However, Yubjha Naap 7.4 semen was slightly alkaline than others. The pH of semen in Bhutanese chicken matches with that of 7.3 the Malaysian local chicken (Malik et al. 2013). 7.2 The pH values of all Bhutanese chicken pH Semen populations were above the semen pH of Nigerian 7.1 78 a a 76 7.0 74 6.9 72 Seim Baylaity Yubjha Naap 70 68 Figure 5: Semen pH among chicken populations b 66 (mean±se of mean). Ns-nonsignificant. 64
Semen motility (%) motility Semen 62 4. CONCLUSIONS Significant variations in semen volume, semen 60 concentration and semen motility exist among all 58 three strains of Bhutanese chicken. Semen color, 56 semen mass activity and semen pH do not differ Seim Baylaity Yubjha Naap significantly. All strains have semen suitable for artificial insemination and cryopreservation. All Figure 4: Semen motility (%) among populations semen characteristics, except semen pH, are (mean±se of mean). Means with different letters positively correlated with secondary sexual traits. differ significantly (p≤0.05). The semen color is independent of all secondary
Table 1: Correlations between secondary sexual traits and semen characteristics. Figures inside cells represent Pearson’s correlation coefficient. Traits Body weight Comb length Comb height Beak length
Semen volume 0.25 0.62* 0.40 0.43 Semen pH -0.55 -0.30 -0.33 -0.23 Semen mass activity 0.52 0.36 0.17 0.09 Semen concentration 0.60* 0.74** 0.74** 0.76** Semen motility 0.54 0.75** 0.57 0.52 *p<0.05, **p<0.01
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Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 7-13, 2018 sexual traits in all three strains of Bhutanese cocks. Dorji N and Dorji P (2015). A country report: To some extent, the secondary sexual traits may be Indigenous chicken farming in Bhutan. Khon used to predict the semen quality. Therefore, the kaen Agrculture Journal, 43 (2): 31-33. cocks with heavier body weight, larger comb size Dorji N and Gyeltshen T (2012). Characterization and longer beak length are recommended to be of family poultry production in Haa and selected as breeder cocks as it has better semen Mongar districts of Bhutan. Livestock Research quality. However, for semen cryopreservation, only for Rural Development, 24 (9). a cock with bigger comb size and longer beak FAO (2014). Family poultry development −issues, length may be selected since heavier cock may not opportunities and constraints. Available at: be suitable as it has low semen pH.
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NBC (2011). Poultry Semen Processing Procedure. reproduction of local barred male chicken of Thimphu. Bhutan. the western highlands of Cameroon: sexual Nidup K and Tshering P (2007). Status of the maturity, fertility and sperm storage term in family poultry production and HPAI in Bhutan. female. Journal of Physiology and Proceedings of the 8th Asian Pacific Poultry Pharmacology Advances. 4 (2): 323-331. Conference of the World’s Poultry Science Tarif AM, Musharraf M, Bhuiyan U, Ferdousy RN, Association. March 2007, Bangkok, Thailand: Juyena NS, and Mollah MBR (2013). 78-83. Evaluation of semen quality among four Peters SO, Shoyebo OD, Ilori BM, Ozoje MO, chicken lines: IOSR Journal of Agriculture and Ikeobi CON, and Adebambo OA (2008). Veterinary Science, 6 (5): 07-13. Semen Quality Traits of Seven Strain of Udeh SOC, Ugwu, Ogagifo NL (2011). Predicting Chickens Raised in the Humid Tropics. semen traits of local and exotic cocks using International Journal of Poultry Science, 7 (10): linear body measurements. Asian Journal of 949-953. Animal Sciences, 5: 268-276. Tabatabaei S, Batavani RA, and Talebi AR (2009). Verge XPXC, Dayer JA, Desjardins RL, and Worth Comparison of semen in indigenous and Ross D (2009). Long-term trends in greenhouse gas broiler breeder rooster. Journal of Animal and emissions from the Canadian poultry industry. veterinary advances, 8 (1): 90-93. The Journal of Applied Poultry Research, 18 Tamang S (2011). Efforts and Initiatives to (2): 210- 222. conserve and promote Indigenous poultry. Zang X, Berry WD, McDaniel GR, Ronald DA, Liu An.GR (Animal Genetic Resources P, Calvert C, and Wilhite R (1999). Body Conservation Program). National Biodiversity weight and semen production of broiler breeder Centre. Indigenous Poultry Conservation males as influenced by crude protein levels and Workshop, Thimphu Bhutan. feeding regimens during rearing. International Tadondjou CDA, Ngoula F, Kana JR, Mube HK, Journal of Poultry Science, 78 (2): 190-196. and Teguia A (2014). Characterization of
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Full length paper Seminal attributes, cooling procedure and post thaw quality of semen of indigenous Khari buck of Nepal
PANKAJ K JHA1, 2*, SAROJ SAPKOTA1, DIL B GURUNG1, RAJU KADEL1, NEENA A GORKHALI1, BHOLA S SHRESTHA1, 3
1Animal Breeding Division, Nepal Agricultural Research Council (NARC), P.O. Box: 1950, Kathmandu, Nepal. 2Department of Surgery and Obstetrics, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh. 3Heifer International, Hattiban, Lalitpur, Nepal; P.O. Box. 6043
*Author for correspondence: [email protected]
Copyright © 2018 Pankaj Kumar Jha. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
Abstract: The objective of the study was to understand the seminal attributes, effectiveness of cooling process and post thawed semen quality of a Nepalese indigenous Khari buck. Two Khari bucks were used for the study. Thirty-two ejaculates (16 ejaculates from each buck) were evaluated for seminal attributes of fresh semen. The mean semen volume, semen color, sperm mass activity, sperm motility, sperm viability, sperm concentration, abnormal acrosome, mid piece and tail, and abnormal head were 0.73 ml, 3.10, 3.80, 80.9%, 94.6%, 2597.0×106 ml-1, 10.7% and 5.00%, respectively. Further, 12 ejaculates (6 ejaculates) from each buck (only having ≥0.5ml semen volume, ≥80% sperm motility, ≥90% sperm viability and ≥2500×106 ml-1 sperm concentration) were considered for cooling, freezing and post thawed semen quality. Tris-egg-yolk- citrate extender was used for semen dilution. The mean sperm motility and viability of post diluted semen after 90 minutes of cooling were 73.8% and 88.1%, respectively. Similarly, the mean sperm motility and viability of post diluted semen after 210 minutes of cooling were 69.2% and 85.0%, respectively. The mean post thaw sperm motility and viability were 49.0% and 81.2%, respectively. The study concluded that the seminal attributes and results of post thaw semen quality are acceptable and indicated the feasibility of cryopreserving Khari buck semen.
Keywords: Artificial insemination; cryopreservation; goat; Khari buck; semen.
1. INTRODUCTION important role in the economy of many peoples. For the goat genetic improvement program, the Goat breeding through artificial insemination (AI) is government has imported some specialized exotic commonly practiced worldwide. It is a very basic goat breeds and semen. Some progress has been tool to speed up genetic improvement and increase made in the initiation of selective breeding within productivity. Goat raising is a popular livestock sub- the local breeds and practice of AI with imported commodity of mixed farming system in Nepal. semen. Importance of AI in goats has drawn more Beside food and nutritional security, it has an 14
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 14-19, 2018 attention among goat farmers. However, importing contained 25 ml of stock solution-II and 50 ml of semen is not a permanent solution at national level; stock solution-III. The stain was left for at least two rather, it is more important to develop and establish weeks for stabilization and maturation. Eosin- its own frozen semen production technique. Semen Nigrosin stain was prepared by dissolving 10 gm quality and its relationship with fertility are said to Nigrosin, 1.7 gm Eosin and 2.9 gm sodium citrate in be a major concern in animal production, hence 100 ml of distilled water. All these reagents and accurate measurement of semen fertilizing potential stains were filtered before use. is of great importance (Grasa et al. 2004). The survivability of sperm cells depends on semen 2.3 Extender preparation quality. Survivality of sperm cells is assessed The Egg Yolk Tris Citrate extender was used for through visual and microscopic evaluation of semen dilution as per Ramukhithi et al. (2011). Egg spermatozoal progressive motility and morphology. Yolk Citrate Tris extender (fraction Part A and B) In Nepal, there is lack of scientific information on was prepared a day before semen collection, using seminal attributes of buck semen, its dilution, the following ingredients: 1.22 gm Tris, 0.68 gm cooling and freezing technique under management citric acid monohydrate, 0.5 gm monohydrate conditions of Nepal. Therefore, a study was glucose, 7 ml glycerol (only in fraction Part B) and conducted with the main objective to evaluate the double distilled water (40 ml in fraction Part A and seminal attributes, cooling procedures and post thaw 33 ml in fraction Part B). After proper mixing, both quality of semen in Indigenous Khari Buck (Capra fraction Part A and B were autoclaved, cooled to hircus) of Nepal. room temperature and stored at 5°C. In the morning, on the day of semen collection, 10 ml fresh hen’s 2. MATERIALS AND METHOD yolk, 50000 IU Penicillin G and 0.05 gm Streptomycin sulfate were mixed in each fraction This study was carried out over a period of four (Part A and B) and maintained at 35°C. months (March to June 2015) at Animal Breeding
Division, Nepal Agricultural Research Council, 2.4 Semen collection and evaluation Kathmandu, Nepal (27.66º N and 85.35 E; altitude Artificial vagina (AV) was used to collect semen as of 1400 meters above sea level). described by Mishra et al. (2010). Bucks were
trained at homosexual mount. The prepuce was 2.1 Animal and management wiped clean with normal saline solution and dried to Two Nepalese indigenous sexually matured healthy reduce contamination before semen collection. Khari bucks aged 2.6 and 2.4 years were selected. Semen was collected after allowing two successive The 2.6-year-old buck weighed 34 kg with body false mounts and transferred into water bath (35°C) condition score 4 and scrotal circumference 27.4 cm. for evaluation. Semen was evaluated according to The 2.4-year-old buck weighed 36 kg with body Jha et al. (2013). Volume was recorded and color condition score 4.5 and scrotal circumference 25.7 was scored visually into 1-4 grades. Sperm mass cm. The bucks were managed under semi-intensive activity was estimated by placing 5µl of fresh semen system. They were allowed for natural grazing on a pre-warmed (35 °C) glass slide and observed during daytime and fed with concentrate at the rate under microscope (40×), without using a cover slip. of 200 gm head-1 day-1. They were vaccinated The mass activity was scored into 1-4 grades. Sperm against foot and mouth disease (FMD), Peste des motility was estimated by placing 5µl of fresh semen Petits Ruminants (PPR) and dewormed with on a pre-warmed (35°C) glass slide and observed Ivermectin twice yearly. under microscope (100×), using a cover slip. Sperm
concentration (106 ml-1) was determined by using 2.2 Reagent and stain preparation hemocytometer technique. The acrosome, mid-piece The reagents and stains were prepared as described and tail abnormalities were examined by wet mount by Jha et al. (2013). Buffered formol saline was technique. A drop (10μl) of diluted fixed semen with prepared by dissolving 6.2 gm disodium hydrogen buffered formal saline (1:100) was placed on clean phosphate, 2.5 gm potassium dihydrogen phosphate, glass slide with a cover slip and examined under 5.4 gm sodium chloride and 175 ml concentrated microscope (1000×). Sperm head morphology was formaldehyde in 1000 ml of distilled water. examined by Williams staining technique. A thin William’s stain was prepared as Stock solution-I by smear of fresh semen (2-4 μl) was prepared, air dissolving 10 gm of basic fuchsin in 100 ml of 95% dried, treated with absolute alcohol for 4 minutes alcohol. Stock solution-II was prepared by and air dried again. Smear was treated with 2% dissolving a saturated solution of bluish eosin in chlormines solution for two minutes, washed in 95% alcohol. Stock solution-III was prepared by distilled water, followed by rinsing in 95% alcohol mixing 10 ml of stock solution-I with 170 ml of 5% and finally stained with carbol fuschsin for eight phenol solution. The final working solution Jha et al. (2018) 15
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 14-19, 2018 minutes. Slide was washed in running tap water, 2.6 Freezing and thawing of spermatozoa dried off and examined under light microscope Freezing of semen straw was done in Styrofoam box (1000×). Sperm viability was estimated by using as described by Ramukhithi et al. (2011). Following eosin-nigrosin stain. A thin semen smear was glycerol equilibration, the semen straws were placed prepared from one large drop (10 μl) of eosin- horizontally on stainless steel wire net and nigrosin stain and one small drop (2 μl) of fresh transferred onto the metal stand in Styrofoam box semen and examined under light microscope (400×). containing liquid nitrogen. The height of the metal Live sperm cells appeared white, unstained against stand was adjusted to 4.5 cm above the level of the purple background of nigrosin, whereas dead and liquid nitrogen. The semen straws were exposed to damaged sperm cells appeared as pink. At least 200 liquid nitrogen vapor for 12 minutes with box top spermatozoa were examined in each smear. closed. Straws were plunged into liquid nitrogen. The straws were collected into goblets and 2.5 Semen dilution and cooling transferred into Cryocan for storage. The post thaw Semen dilution and cooling was performed as sperm motility and viability were examined. described by Naing et al. (2011). Only ejaculations Thawing procedure was carried out in a water bath having ≥0.5ml semen volume, ≥80% sperm motility, at 37°C for 20 seconds. Data were analyzed ≥90% sperm viability and ≥2500×106 ml-1 sperm statistically for mean and standard deviation. Data concentration were considered for semen dilution. analysis was carried out with the statistical software Semen dilution was performed by two step dilution SPSS version 20. technique to obtain a final concentration of 100×106 per 0.25 ml French mini semen straw. Soon after 3. RESULTS evaluation, calculated volume of fraction Part A was 3.1 Seminal attributes of fresh semen mixed with semen. Diluted semen with fraction Part A total of 32 ejaculates (16 ejaculates from each A and calculated volume of fraction Part B were Khari buck) were evaluated for seminal attributes. transferred for cooling. After 90 minutes, the The results are presented in Table 1. calculated volume of fraction Part B was mixed. The final diluted semen suspension was loaded into 0.25 3.2 Post dilution semen quality ml semen straws with the help of micropipette and A total of 12 ejaculates (six ejaculates from each sealed with poly vinyl chloride (PVC) powder. Khari buck) were used. The results of post-dilution Sealed straws were left further for 120 minutes for motility and viability of sperm cells during cooling glycerol equilibration at 5°C. Semen samples were at 5°C are presented in Table 2. After 90 minutes evaluated for sperm motility and viability after 90 post dilution, the mean sperm motility and viability minutes and 210 minutes of cooling. were 78.8±4.8% and 88.1±2.6%, respectively. After Table 1: Seminal attributes of fresh semen of Nepalese indigenous Khari bucks (Mean±SD) Buck ID Volume Color Mass Motility Viability Concentration Abnormal Abnormal 6 -1 (ml) (1-4 activity (%) (%) (×10 ml ) acrosome, head (%) grade) (1-4 mid-piece grade) & tail (%)
Khari 1 (n=16) 0.8±0.3 3.2±0.4 3.7±0.5 83±4.7 95±2.6 2576±469 10.3±1.6 4.6±1.7 Khari 2 (n=16) 0.6±0.3 3.1±0.3 3.9±0.3 78±5.4 94±1.2 2618±347 11.1±1.9 5.4±1.6 Pooled (n=32) 0.7±0.3 3.1±0.3 3.8±0.4 81±5.6 95±2.0 2597±407 10.7±1.8 5±1.7 1= Watery; 2= Yellowish white; 3= Milky white; 4= Creamy white. Sperm mass activity (scored into 1- 4 grades): 1= No mass activity; 2= Slow wave motion without forming any waves; 3= Rapid wave motion with formation of eddies at the end of waves; 4= Very rapid wave motion with distinct with distinct eddies.
Table 2: Post-dilution motility and viability of sperm cells at 5°C (Mean±SD) Semen + fraction Part A Semen + fraction Part A + fraction Part B (after 210 minutes) (after 90 minutes) Buck ID Motility (%) Viability (%) Motility (%) Viability (%) Khari 1 (n=6) 77.5±2.7 89.0±3.0 73.3±4.1 85.5±1.8 Khari 2 (n=6) 70.0±3.2 87.2±2.0 65.0±4.5 84.5±1.5 Pooled (n=12) 73.8±4.8 88.1±2.6 69.2±6.0 85.0±1.7
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210 minutes post dilution, the mean sperm motility Khari buck was within the range of 2619.58 to and viability were 69.2±6.0% and 85.0±1.7%, 2988±9.6×106 ml-1 found in Black Bengal buck respectively. The motility and viability percent (Faruque et al. 2007). In contrast, Shamsuddin et al. decreased slightly with the advancement of cooling (2002) reported higher sperm concentration time. 4187±1768 to 7990±2360 ×109 ml-1 in Bangladeshi buck. The standard sperm concentration of a good 3.3 Post thaw semen quality buck is ≥ 2×109 ml-1 (Paulenz et al. 2005; Hidalgo et The results of post-thawed motility and viability of al. 2007). The abnormalities of sperm morphology sperm cells are presented in Table 3. The mean of Khari buck agree with that of Ghalsasi (2004) values of post thawed motility and viability of sperm who suggested abnormal sperm acrosome, mid- cells were 49.0±4.6 and 82.7±2.3%, respectively. piece and tail below 15% and, abnormal sperm head below 5%, as good enough for AI. The variation in Table 3: Post-thawed sperm motility and viability seminal attributes might be due to type of breed, age of sperm cells (Mean±SD) of buck, season, physiological status and genetics of Buck ID Motility (%) Viability (%) buck (Webb et al. 2004; Daramola et al. 2007; David et al. 2007). The feeding regime, method of Khari 1 52.0±2.7 84.2±1.5 collection and frequency of collection also affect Khari 2 46.0±4.2 81.2±1.9 semen quality (Jimenez et al. 2005; Yamashiro et al. Pooled 49.0±4.6 82.7±2.3 2006; Sundararaman et al. 2007; Zarazaga et al. 2009).
4. DISCUSSION 4.2 Post dilution semen quality In our study, the slight decrease in sperm motility of Desired fertility is the main goal of any breeding Khari buck after 90 and 210 minutes of post dilution program. Adequate sperm functional like normal at 5°C agrees with the findings of Shamsuddin et al. morphology, motility and ability to undergo the (2002) who reported sperm motility 70.00±2.89 to events of capacitation are necessary for successful 73.33±1.67%. Similarly, the sperm viability of Khari pregnancy. buck after 90 and 210 minutes of post dilution at 5°C
also agrees with the findings of Samsuddin and 4.1 Seminal attributes of fresh semen Chanda (1998) who found 84.4±4.0% live In this study, the semen volume of Khari is in spermatozoa. The decrease in percentage of motile agreement with Khandoker et al. (2006) and and viable sperm with advancement of preservation Ramukhithi et al. (2011) who reported 0.7±0.08 to time at 5°C is probably due to cold shock 1.57±0.35 ml in South African indigenous buck and experienced by the spermatozoa (Upreti et al. 1997). 0.43±0.03 to 0.45±0.22 ml in Black Bengal buck. In The cold shock of spermatozoa is associated with contrast, Webb et al. (2004) and Choe et al. (2006) oxidative stress induced by reactive oxygen species reported semen volume 1.57±0.35 to 1.77±0.3 ml in generation (Gadea et al. 2005). different buck breeds. The acceptable semen volume for buck ranges from 0.5 to 2.0 ml (Al- Ghalban et 4.3 Post thaw semen quality al. 2004; David et al. 2007). The semen color of The motility and viability of sperm cells decreased Khari was consistently milky white (ranged 3-4). after post thawing. The post thawed sperm motility The normal semen color of buck is yellowish-white. is in agreement with the results of Ramukhithi et al. The color pattern of the neat semen is species (2011) who reported sperm motility of 49.3±3.4%. specific, dependent on sperm concentration and Sperm motility decreases up to 30% after post presence of pigmented proteins and carotenoids in freezing thawing, which is assumed to be acceptable the seminal plasma (Hafez 1987). The sperm mass (Kozdrowski et al. 2007). However, the bench mark activity of Khari buck recorded rapid to very rapid of post thaw sperm motility in buck is above 50% wave motion with distinct eddies formation. Almost (Biswas et al. 2002). The post thawed sperm motility similar range was observed by Ferdinand et al. obtained in this study is lower than the bench mark. (2002) and Shamsuddin et al. (2002). The sperm The quality of frozen semen decreased after thawing. motility of Khari is similar to that of Shamsuddin et During freezing and thawing process, the metabolic al. (2002) who observed sperm motility 76.0±1.45 to activity of spermatozoa is arrested and spermatozoa 78.00±1.11% and 72.9±6.7 to 80.3±3.0% in fresh undergoes several ultrastructural, biochemical and buck semen. The acceptable sperm motility of fresh functional changes. These changes are also buck semen is above 70% (Nur et al. 2005). In this associated with the temperature of freezing and study, the sperm viability of Khari buck is higher thawing process (Maxwell and Salamon 1993; than 70.0-90.0% reported for different goat breeds Dorado et al. 2007). (Igboeli 1974). The sperm cell concentration of Jha et al. (2018) 17
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5. CONCLUSION buck age, storage duration, storage temperature and diluent of fresh West African Dwarf buck The seminal attributes and results of post thaw semen. Journal of Reproduction and Infertility, semen quality were satisfactory and in accordance 3: 58-62. with works carried out in other countries, which Gadea J, Garcia-Vazquez F, Matas C, Gardon JC, indicates the feasibility of cryopreserving buck Canovas S, and Gumbao D (2005). Cooling and semen. For more validation, the study recommends freezing of Boar spermatozoa: Supplementation research with large number of bucks, different types of the freezing media with reduced glutathione of diluents and freezing trials by removing seminal preserves sperm function. Journal of Andrology, plasma, followed by pregnancy rate. 2: 396-404.
Ghalsasi PM (2004). Technical training manual on Acknowledgements artificial insemination in goats. Maharashtra The authors wish to acknowledge the Nepal Goat and Sheep Research and Development Agricultural Research Council (NARC) for financial Institute, Phlatan Nimbkar Agricultural support, and providing animal shed and laboratory Research Institute, Phlatan, India. facilities. Grasa P, Pe-Perez R, Baguena O, Forcada F, Abecia A, Cebrian-Perez JA, and Muino-Blanco T REFERENCES (2004). Ram sperm selection by a dextran/swim- Al-Ghalban AM, Tabbaa MJ, and Kridli R (2004). up procedure increases fertilization rates Factors affecting semen characteristics and following intrauterine insemination in scrotal circumference in Damascus bucks. Small superovulated ewes. Journal of Andrology, 25: Ruminant Research, 53: 141-149. 982-990. Hafez ESE (1987). Reproduction in Farm Animals: Biswas D, Bari FY, Shamsuddin M, Rahman MM, th and Rahman MM (2002). Determination of 315-481. 5 edition. Lea and Febiger, glycerol percentages for preserving the Black Philadelphia, USA. Bengal buck (Capra hircus) spermatozoa for Hidalgo M, Rodriguez I, and Dorado J (2006). long time. Pakistan Journal of Biological Influence of staining and sampling procedures Sciences, 5: 715-718. on goat sperm morphometry using the Sperm Choe CY, Kim JG, Cho SR, Son DO, Young KK, Class Analyzer. Theriogenology, 66: 996-1003. Balasubramanian S, Choe SY, and Rho GJ Igboli G (1974). A comparative study of the semen (2006). Influence of seasons, extenders, slow and seminal characteristics of two breeds of and rapid freezing on seminal characters in goats. East African Agricultural and Forestry Koeran Native bucks. Reproduction in Domestic Journal, 40:132-137. Animals, 41: 55-60. Jha PK, Paul AK, Rahman MB, Tanjim M, Bari FY, Daramola JO, Adeloye AA, Fatoba TA, and and Alam MGS (2013). Improvement of Soladoye AO (2007). Induction of puberty in Preservation Quality of Chilled Bull Semen West African Dwarf buck-kids with exogenous Using α-tocopherol as an Antioxidant. Journal melatonin. Livestock Research for Rural of Embryo Transfer, 28: 31-39. Development, 19: 9. Jimenez F, Puchades S, Gadea J, Vicente JS, and David I, Druart X, Lagriffoul G, Manfredi E, Robert- Viudes-de-Castro MP (2005). Effect of semen Granie C, and Bodin L (2007). Genetic and collection method on pre- and post-thaw Guirra environmental effects on semen traits in ram spermatozoa. Theriogenology, 64: 1756- Lacaune and Manech tete rousse AI rams. 1765. Genetics Selection Evolution, 39: 405-419. Khandoker MAMY, Afroz S, Islam MR, and Husain Dorado J, Rodriguez I, and Hidalgo M (2007). SS (2006). Cryopreservation of buck semen. In: Cryopreservation of goat spermatozoa: proceedings of the XII AAAP Animal Science Comparison of two freezing extenders based on Congress, Bexco, Busan, Korea: 18-22. post-thaw sperm quality and fertility rates after Kozdrowski R, Dubiel A, Bielas W, and Dzieciol M artificial insemination. Theriogenology, 68: (2007). Two protocols of cryopreservation of 168-177. goat semen with the use of computer-assisted Faruque MH, Bari FY, Siddiqui MAR, and semen analysis system. Acta Veterinaria Brno, Shamsuddin M (2007). Fertilizing capacity of 76: 601-604. buck (Capra hircus) semen frozen with different Maxwell WM and Salamon S (1993). Liquid storage concentrations of egg yolk. Journal of the of ram semen: A review. Reproduction Fertility Bangladesh Agricultural University, 5: 95-104. and Development, 5: 613-638. Ferdinand N, Thomas TT, Augustave K, Henry DF, Mishra B, Alam MGS, Khandokar MAMY, Fernand T, and Etienne PT (2002). Effects of Mazumder S, and Munsi MN (2010). Qualities Jha et al. (2018) 18
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of goat semen in Tris-Citrate-Glucose extender regard to ejaculate numbers, collection intervals, containing glutathione. Bangladesh diluents and preservation periods. Reproduction Veterinarian, 27: 46-55. in Domestic Animals, 35: 53-57. Naing SW, Haron AW, Khan MA, and San MM Sundararaman MN, Kalatharan J, and Edwin MJ (2011). Effect of seminal plasma removal, (2007). Attempts to achieve semen collections washing solutions, and centrifugation regimes from incapacitated Boer bucks by electro- on Boer Goat semen cryopreservation. Pertanika ejaculation. Asian Journal of Animal and Journal of Tropical Agricultural Science, 34: Veterinary Advances, 2: 244-246. 271-279. Upreti GC, Jensen K, Oliver JE, Duganzich DM, Nur Z, Dogan I, Gunay U, and Soylu KM (2005). Munday R, and Smith JF (1997). Motility of ram Relationships between sperm membrane spermatozoa during storage in a chemically- integrity and other semen quality characteristics defined diluent containing antioxidants. Animal of the semen of Saanen goat bucks. Bulletin of Reproduction Science, 48: 269-278. the Veterinary Institute in Pulawy, 49: 183-187. Webb EC, Dombo MH, and Roets M (2004). Paulenz H, Soltun K, Adnoy T, Andersen K, and Seasonal variation in semen quality of Gorno Soderquist L (2005). Effect of different Altai cashmere goats and South African extenders on sperm viability of buck semen indigenous goats. South African Society for stored at room temperature. Small Ruminant Animal Science, 34: 240-243. Research, 59: 89-94. Yamashiro H, Kumamoto K, Wang H, Yamashita Y, Ramukhithi FV, Nedambale TL, Sutherland B, and and Terada T (2006). Effect of semen collection Lehloenya KC (2011). Cryopreservation of in extender solution on the characteristics of South African indigenous goat semen. African goat spermatozoa. Journal of Reproduction and Journal of Biotechnology, 10: 17898-17902. Development, 52: 397-406. Shamsuddin M and Chanda PK (1998). Effects of Zarazaga LA, Guzman JL, Dominguez C, Perez MC, diluents and dilution rates on the preserved buck and Prieto R (2009). Effects of season and semen. In: 4th Asian Symposium on Animal feeding level on reproductive activity and semen Biotechnology. Kamiina, Japan: 177-181 quality in Payoya buck goats. Theriogenology, Shamsuddin M, Amiri Y, and Bhuiyan MMU 71: 1316-1325. (2002). Characteristics of buck semen with
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Full length paper Length-weight relationship and relative condition factor of Golden Mahseer (Tor putitora) under pond-reared condition in southern foothills of Bhutan
NAMGAY DORJI*, DRUKPOLA, JAMYANG NORBU AND NETEN
National Research and Development Centre for Aquaculture, Department of Livestock, Ministry of Agriculture and Forests, Gelephu, Sarpang, Bhutan. *Author for correspondence: [email protected] Copyright © 2018 Namgay Dorji. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: A study was conducted with the objective to establish the first ever length- weight relationship and evaluate the physical robustness of the endangered Golden Mahseer (Tor putitora Hamilton 1822) in Bhutan. The study samples were from populations reared in fertilized earthen ponds at National Research and Development Centre for Aquaculture (NRDCA), Gelephu. Data on total length and total weight of 120 juvenile and adult specimens of fish were collected in November, 2017. To enable simple linear regression analysis for estimating the coefficient of total length in describing total weight, the linear growth model (ln W = ln a+b. ln L) was used. An indicator of physical robustness was estimated using Le Cren’s formula for relative W condition factor (Kn) (Kn = ). Length-weight relationships and relative condition Ŵ factors were estimated separately for two different groups of juveniles, adult males, adult females and pooled data. The relationships between total weight and total length across groups and for the pooled data were highly significant at 99% significance level. The length-weight relationships and relative condition factors (mean values) were: ln W= -2.581+1.749 ln L and 1.044 for 9-month-old juveniles; ln W= -4.062+2.782 ln L and 1.025 for 13-month-old juveniles; ln W= -4.385+2.946 ln L and 1.066 for adult males; ln W= -5.420+3.195 ln L and 1.113 for adult females; and ln W=-3.979+2.822 ln L and 1.064 for pooled data.
Keywords: Golden Mahseer; length-weight relationship; pond; relative condition factor.
1. INTRODUCTION the waters of the Himalaya and south Asia, in countries like India, Nepal, Pakistan, Bhutan and Golden Mahseer (Tor putitora Hamilton 1822) is a Myanmar. The International Union for freshwater fish belonging to the family Cyprinidae. Conservation of Nature (IUCN) evaluates Golden It is reported to grow to a maximum size of 2.75 m Mahseer as an endangered fish. IUCN reports that length and 54 kg weight (wikipedia). Also, the wild population of this fish has already declined commonly called as the Putitor Mahseer and by 50% and that it may decline further by even up Himalayan Mahseer, the Golden Mahseer is an to 80% as a result of habitat destruction and excellent game-fish, actively sought by recreational reproductive cycle disruptions caused by fishing enthusiasts for its remarkable fighting anthropogenic activities such as hydroelectric ability. It is also a prized food-fish. It is found in 20
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 20-24, 2018 power dam construction. There is an urgent need to 2. MATERIALS AND METHOD initiate efforts to conserve Golden Mahseer to 2.1 Study site and samples prevent it from becoming extinct in several The study was conducted at NDRCA (26o52’7.68” locations across the range of its distribution (IUCN N and 9o29’45.25”), Gelephu, Bhutan. The total 2013). length to the nearest centimeter (cm) and total Golden Mahseer occurs in several water bodies weight to the nearest gram (g) of 120 Golden in Bhutan. It is generally considered to be the most Mahseer were measured with a Vernier caliper and important fish species of the country’s aquatic a high-precision digital weighing balance, ichthyofauna, outclassing all other fish species in respectively, in November, 2017. The study sample terms of appearance and size. Major rivers like comprised 30 adult males, 30 adult females, 30 9- Punatshangchhu and Mangdechhu have month-old and 30 13-month-old juveniles. The traditionally been considered safe haven for Golden adult fish were collected from Mangdechhu in 2014 Mahseer. However, in recent years, with while the juveniles were the offspring of these hydropower facilities being constructed on these adults produced at NRDCA. The fish were fed rivers, it is believed that the fish’s traditional routes supplementary diet on a daily basis. for spawning migration therein and breeding and nursing grounds upstream have been undermined 2.2 Computation of LWR and Relative condition significantly. factor (Kn) This paper examines the Length-Weight To establish LWR, separately for adult males, adult Relationship (LWR) and relative condition factor of females, 9-month-old juveniles and 13-month-old Golden Mahseer reared with daily supplementary juveniles and for pooled data, the allometric growth feeding at 2% of total stock weight in fertilized model was used was: W = aLb, where W= weight, earthen ponds at the NRDCA, Gelephu, Bhutan. L= length, a= constant and b= exponent (Le Cren LWR is an important biometric indicator that has 1951). gainful uses in fisheries management and research. The allometric growth equation was log- It quantifies the relationship between the weight transformed using natural logarithm to derive the and length of fish and can therefore be used to linear equation model: ln W = ln a +b. ln L (a and estimate the weight of a fish corresponding to its b were estimated by least square method). length. For example, the length observations from To estimate the Relative Condition Factor (Kn), underwater visual census methods can be converted group-wise and for pooled data as with the LWR, into weight estimates for biomass estimation Le Cren’s relative condition factor was computed (Froese 1998). LWR also enables the measurement with the following equation. Relative condition of the variation from the expected weight for length factors (Kn) of individual fishes within the context of individual fish or groups of fish as an indication of the groups they belong and in the context of the of fatness, general wellbeing, gonadal development pooled data were calculated using their observed etc. (Le Cren 1951). LWR also has other important lengths and corresponding LWR equations. applications, such as to set yield equations W Kn = (where W= observed weight and Ŵ= (Beverton and Holt 1957). Several past studies have Ŵ described the LWRs and the general wellbeing of standard weight calculated using the estimated Golden Mahseer from different waters (Islam et al. LWR). 2002; Johal et al. 2005; Atkore et al. 2007; The dataset was analyzed with the statistical Gandotri et al. 2008; Patiyal et al. 2013; Rawal et software Stata Version 13. al. 2013; Ali et al. 2014; Khajuria et al. 2014; Naeem et al. 2014). 3. RESULTS AND DISCUSSION Relative condition factor is a robust indicator of 3.1 LWR the general wellbeing (condition) of fish. the Table 1 presents the total length and total weight relative condition factor enables distinguishing for groups and pooled data. While the smallest fish between and measuring separately the influences on recorded during the study was 2.3 cm long and 0.32 condition of length and other factors such as g heavy, the largest recorded was 68.3 cm long and environment, food supply and degree of 3760 g heavy. The curvilinear relation between the parasitization (Le Cren 1951). total weight (TW) and total length (TL) is presented However, to date, no such studies have ever in Figure 1. The linear relationship between log- been conducted on Golden Mahseer occurring in transformed total weight (lnTW) and log- the waters of Bhutan. The present study is the first transformed total length (lnTL) is presented in ever analysis of LWR and relative condition factor Figure 2. Figure 3 presents the scatterplot of the of Golden Mahseer in Bhutan. residuals from regressing lnTW on lnTL against
lnTL, which showed no visible pattern to how the Dorji et al. (2018) 21
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Table 1: Total Length and Total Weight of sample groups. Sample group Total Length (cm) Total Weight (g) β Min Max Mean SDβ Min Max Mean SD 00.32 01.99 00.73 00.35 9-month-old juveniles 2.30 05.20 03.52 00.62 13-month-old juveniles 12.10 27.0 19.52 03.71 16.00 178.0 73.40 40.64 Adult males 41.00 54.60 48.30 3.336 696.00 1672.0 1162.13 249.53 Adult females 48.30 68.30 56.75 04.76 1076.0 3760.0 1835.00 553.70 Pooled data (N=120) 2.30 68.30 32.04 21.85 00.32 3760.0 767.91 828.68 βStandard Deviation residual varied with lnTL, thus, indicating that the values were 1.066, 1.113, 1.044, 1.025 and 1.064 model used in this study was correct. for adult males, adult females, 9-month-old The results of regressing lnTW on lnTL juveniles, 13-month-old juveniles and pooled data, separately for adult males, adult females, 9-month- respectively, which indicates that on average, old juveniles and 13-month-old juveniles, and for Golden Mahseer at NRDCA are in good physical pooled data are presented in Table 2. The condition. The supplementary diet provided to fish mathematical forms of group-wise and pooled data on a daily basis assured an environment with LWRs, derived using the estimates for a and b are abundant food supply, which is reflected by proper presented in Table 3. growth and gain in physical mass of fish. Further, Across groups and for the pooled data, the total because the fish population was reared in length of Golden Mahseer was highly significant in confinement, its movement was substantially explaining the variances in its weight. Except in the restricted, which may have enabled it to accumulate case of 9-month-old juveniles, the value of b for all additional physical mass, compared to a population other groups was within 2.5 to 4, thus, conforming of comparable attributes in the wild where to the observations of Hile and Martin (as cited in movement is not restricted and the fish has to spend Le Cren 1951). The value of b was close to 3 for a considerable amount of energy on foraging. The adult males and females, 13-month-old juveniles Golden Mahseer’s satisfactory physical condition at and the pooled data suggests that Golden Mahseer NRDCA may partly be attributed to the water being at NRDCA grows isometrically (where b=3) and maintained at optimum quality in terms of content equally in all directions as per Spencer’s Cube law of dissolved oxygen, pH, alkalinity, carbon dioxide, (as cited in Froese 2006). Our findings agree with etc., and the fish being protected from undue stress those reported by several authors (Islam et al. 2002; from illegal fishing activities and other factors that Johal et al. 2005; Gandotra et al. 2008; Rawal et al. routinely disturb the comparable populations in the 2013; Ali et al. 2014; Khajuria et al. 2014). wild. However, for the 9-month-old juveniles sample, b For the 9-month-old juveniles, the Kn value of was estimated to be 1.749, which is outside the 1.044 indicated a negative allometric growth range observed by Hile and Martin (as cited in Le pattern, which could possibly be due to natural Cren 1951). This is consistent with the findings growth pattern. The Kn values of this study are reported for Golden Mahseer juveniles in India similar to those reported by Islam et al. (2002) for (Atkore et al. 2007; Khajuria et al. 2014). A pond-reared Golden Mahseer. negative allometric growth of 0+ year old Golden Mahseer was reported by Gandotra et al. (2008). In 4. CONCLUSION this study, the negative allometric growth observed This study established separately the first ever for juveniles could be explained from two fronts. LWRs for adult males, adult females, two The negative allometric growth is probably due to independent groups of juveniles and pooled sample the fish’s natural growth pattern where weight does of the endangered Golden Mahseer in Bhutan. In not increase as much as length. It could also be due spite of the samples being derived from pond- to unsatisfactory growth of fish, resulting from reared populations, the findings of this study should inadequate food, parasitic infection, disease and serve as baseline for further similar studies in the unsuitable environmental conditions or a country, particularly for Golden Mahseer combination of these factors. populations in Mangdechhu river basin.
The physically robust population of Golden 3.2 Relative Condition Factor (Kn) Mahseer should enable its use as benchmarks in The means of Kn for different groups of Golden assessing the robustness of cultivated and wild Mahseer are presented in Table 4. The Mean Kn populations of Golden Mahseer.
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However, the sample size in this study is rather Table 2: Coefficient estimates from regressing small and it may not have represented the natural log of Total Weight on natural log of Total population adequately. Therefore, further studies Length for similar groups of Golden Mahseer with larger Sample Constant ln (Total R2 sample sizes and randomized sampling procedures Group Length) are recommended for comparison with the findings of the present study. 9-month-old -2.581*** 1.749*** 0.545 juveniles (0.378) (0.301) Total Weight VS Total Length for Pooled data 13-month- -4.062*** 2.782*** 0.961 old juveniles (0.312) (0.105)
4000 Adult males -4.385*** 2.946*** 0.848 (0.911) (0.235)
3000 Adult -5.420*** 3.195*** 0.898 females (0.820) (0.203) Pooled data -3.979*** 2.822*** 0.995
2000 (N=120) (0.057) (0.017) TotalWeight (g) Standard errors are reported in parentheses. 1000 ***p≤0.001.
0
0 20 40 60 80 Table 3: Length-Weight relationships derived Total Length (cm) using coefficient estimates from regressing natural
Figure 1: Scatterplot for pooled data of natural log log of Total Weight on natural log of Total Length of total weight against log of total length. Regression Equation Allometric Sample (ln W=ln a +b. ln L) Growth lnTotal Weight VS lnTotal Length for Pooled data Group Equation
8 (W = aLb)
2.94 6 Adult males lnW=-4.385+2.946 W=0.012 L
lnL 4
Adult lnW=-420+3.195 lnL W=0.004 L3.20 lnTW
2 females 9-month-old lnW=-2.581+1.749 W=0.075 L1.75 0 juveniles lnL 2.78 -2 13-month- lnW=-4.062+2.782 W=0.017 L 1 2 3 4 lnTL old lnL juveniles Figure 2: Scatterplot for pooled data of total weight against total length. Pooled data lnW=-3.979 +2.822 W=0.018 2.822 lnL L Residuals VS lnTotal Length for Pooled data Acknowledgements
.5 The authors are highly grateful to Department of Livestock (DoL), Ministry of Agriculture and Forests (MoAF), Royal Government of Bhutan 0 (RGoB) for providing support and funds for the study. The authors also would like to profusely Residuals thank the staff and field attendants of NRDCA for -.5 enabling the study by contributing to data collection in a substantial manner.
-1 EFERENCES 1 2 3 4 R lnTL Ali S, Barat A, Kumar P, Sati J, Kumar R and Figure 3: Scatterplot for pooled data of residuals Haldar RS (2014). Study of the length-weight against natural log of total length. relationship and condition factor Tor putitora, Golden Mahseer from Himalayan rivers of Dorji et al. (2018) 23
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Table 4: Descriptive statistics of group-wise and pooled relative condition factors. Relative Condition Factor (Kn) Group n Min. Max. Mean SDβ
1. Adult males 30 0.878 1.299 1.066 0.093 2. Adult females 30 0.859 1.332 1.113 0.100 3. 9-month-old juveniles 30 0.497 1.502 1.044 0.260 4. 13-month-old juveniles 30 0.714 1.215 1.025 0.104 Pooled data 120 0.455 1.926 1.064 0.228 βStandard Deviation
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Full length paper Effect of stocking density on performance of cultivable carps in Southern Bhutan
PEMA THINLEY*, DRUKPOLA AND NAMGAY DORJI
1National Research and Development Centre for Aquaculture, Department of Livestock, Ministry of Agriculture and Forests, Gelephu, Sarpang, Bhutan.
*Author for correspondence: [email protected]
Copyright © 2018 Pema Thinley. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: The effect of different stocking rates on growth performance of two fish species namely Rohu (Labeo rohita) (Hamilton 1882) and Cauvery white carp (Cirrhinus mrigala) (Hamilton 1822) was evaluated in 180 days of culture period. Four different treatments were evaluated. The treatments were stocking rates of two fish m-2 as T1, four fish m-2 as T2, six fish m-2 as T3 and eight fish m-2 as T4. The experiment was conducted in National Research and Development Centre for Aquaculture, Gelephu, Bhutan. All other management aspects such as feeding, manuring, fertilization and liming were same for all treatment ponds. The initial average weight and length of Rohu was 8.10 g and 8.74 cm, respectively, whereas the initial average weight and length of Cauvery white carp was 7.49 g and 9.20 cm, respectively. Fishes were fed @ 5% of body weight for three times a day: morning at 8.00 am, afternoon at 12.00 pm and evening at 5.00 pm. Fish growth parameters were recorded monthly through sampling 30 fish of each species from each treatment pond. Although, there was no significant difference among pond parameters, the final fish body weight was significantly greater for the stocking rate of four fish m-2. The findings suggest that stocking rate of four fish m-2 is most appropriate in achieving optimum growth of Rohu and Cauvery white carp in subtropical region of Bhutan.
Keywords: Cauvery white carp; Feed Conversion Ratio; Growth; Rohu, Stocking rate; Specific Growth Rate.
1. INTRODUCTION in six months with an average individual harvest weight of 0.70 kg. But the current average Fish is an important meat commodity in diet of productivity of Bhutanese fish farm is about 1.4 Bhutanese people. Fish is a good source of protein, MT and the average individual harvest weight is fatty acids, vitamins, minerals and essential 0.23 kg. Fish farmers in Bhutan often face low micronutrients. Realizing the nutritional importance yield that culminates in low profit margins. Fish of fish, aquaculture was initiated in early 1980s in growth is affected by several factors. Of these Bhutan. Over the last three decades, fish farming factors, stocking rate is important. The poor growth has expanded and aquaculture is becoming a of fish, to some extent arises from lack of sufficient profitable enterprise. However, the average knowledge on the best practice of fish stocking Bhutanese fish farm’s productivity is low due to densities in ponds. For this reason, a study was poor culture management practices. Under proposed to address the concern in a local setting optimum condition, generally, one acre of fish farm that could be representative of various fish farm is capable of producing 4.0 metric ton (MT) of fish 25
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 26-30, 2018 sites in Bhutan. In particular, a study on the effects mortality was assessed by transferring at least 30 of different stocking rate on growth performance of individuals in hapa for three days in all treatments. Rohu (Labeo rohita Hamilton) and Cauvery white carp (Cirrhinus mrigala Hamilton) has not been 2.3 Stocking conducted. Therefore, there is a need for a fish The research ethics guidelines of Department of production technology capable of producing an Livestock were followed and all fish were average individual harvest weight of minimum subjected to minimal stress and pain during the 0.650 kg in six months, which is close to optimum entire study period. Each treatment pond was productivity. stocked with stunted fingerlings on 30th March, The research was envisaged to generate 2017 two weeks after manuring or after the ponds knowledge on appropriate fish stocking density that were productive. The initial average weight and would maximize production and profitability of length of Rohu were 8.10 g and 8.74 cm, aquaculture enterprises in subtropical region of respectively. The initial average weight and length Bhutan. We conducted an on-farm study with the of Cauvery white carp were 7.49 g and 9.20 cm, objective to establish the most appropriate stocking respectively. Single Stocking Single Harvesting densities for Rohu and Cauvery white carp in (SSSH) culture system with combination of two earthen ponds. This was achieved by evaluating the species (consisting of two Indian major carp effects of four different stocking rates on growth species) was followed. Stocking species ratio was performance of Rohu and Cauvery white carp 6:4, where 60% were Rohu and 40% were Cauvery under Bhutanese management conditions. white carp. The fingerlings were conditioned at least for 24 hours to adapt them to practical 2. MATERIALS AND METHODS condition associated with live fish transportation. Stocking rate was the treatment factor. 2.1 Pond preparation
Four small earthen ponds at NRDCA were used for 2.4 Feeding on-farm trial as treatment ponds. Each pond size Considering the availability of local feed was 270 m2. Considering the limited ponds at ingredients, crude feed formulated from rice bran NRDCA, there was no replication of ponds and and mustard oil cake in 2:3 ratios (40%:60%) was treatments. The ponds were thoroughly prepared used in all treatments. The fish was provided with and limed @ 250 kg ha-1 application-1 (Das et al. feed @ 5% of body mass per day and feeding rate 2011). Bamboos along with its twigs were provided was adjusted to 2% during the course of research to all the treatment ponds so that it can serve as based on feed utilization (Jena et al. 2001). The hideouts from predators and also serve as substrate biomass of fish was estimated every month based for periphyton production. All ponds were provided on sampling data. Feeding was done three times a with four numbers of bamboo twigs of day as feeding frequency of more than three times approximately same surface area. Organic manure has shown significant growth as compared to two in the form of cattle dung was applied as basal dose times in case of Rohu (Abid and Ahmed 2009). @ 4 tones ha-1 yr-1. Manure and fertilizer were Feeding was done at 8 am in the morning, 12 pm in applied three times to all treatment ponds during the afternoon and 5 pm in the evening. Same the entire culture period, based on the planktonic feeding methods were followed for all treatments. productivity of individual ponds, which was Feed was dispensed along the four corners of pond evaluated on weekly basis. The thumb rule of in the form of ball. manuring is to ensure optimum plankton bloom at any given point of time. Manuring was done one 2.5 Other Management Aspects week after liming in all treatment ponds. The water flow inside the ponds was adjusted to
maintain the optimum water level in fish pond by 2.2 Experimental design and treatments compensating the water loss through seepage and The experimental design was Randomized evaporation. In order to maintain desired water pH Complete Block with one treatment factor, namely and pond hygiene, lime was applied @ 100 kg ha-1 four stocking densities. The fish species were Rohu application-1 once in three months. Apart from this, and Cauvery white carp. The stocking densities the lime was applied as and when required during were two fingerlings per square meter for treatment the course of culture period. 1 (T ), four fingerlings per square meter for 1 Manuring was done with cow dung on the basis treatment 2 (T ), six fingerlings per square meter 2 of water transparency to ensure optimum plankton for treatment 3 (T ) and eight fingerlings per square 3 population at any given point of time. The meter for treatment 4 (T ). In total, the initial 4 transparency of all ponds was judged visually, stocking densities of T , T , T and T ponds were 1 2 3 4 supplemented by elbow method on weekly basis or 534, 1068, 1620 and 2160, respectively. Delayed Thinley et al. (2018) 27
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 26-30, 2018 as and when required to decide on manuring. Key used to analyze data. water quality parameters such pH, DO2, temperature, transparency and color were recorded 3. RESULTS AND DISCUSSION during the entire culture period. 3.1 Water quality parameters
There were no significant differences in water 2.6 Duration of experiment quality parameters among the treatments (Table 1). The trial was conducted for a period of six months. However, when compared between treatments, In order to assess the growth and health of the fish, highest pH was recorded in T2, followed by T1, T4, sampling was done at monthly interval. Minimum and then T3 being the lowest. T1 exhibited highest of 30 individuals of each species was considered as DO value, followed by T2, T3 and T4, an effective sample size. 2 respectively. The highest and lowest temperatures
was recorded in T2 and T1, respectively. However, 2.7 Data collection and analysis all the parameters recorded were within the optimal Fish was sampled and the length and weight were range that is ambient for normal growth recorded at monthly interval, using measurement performance of Rohu and Cauvery white carp as scale and electronic balance. The following reported by Hossain (2000). Similar result is also equations were used to evaluate the growth reported by Haque et al. (2015) on experiment performance of fish under different treatments. conducted on different stocking densities in i. Mean gain in length (cm) = Mean final length Bangladesh. (cm) – Mean initial length (cm) ii. Mean gain in weight (g) = Mean final weight 3.2 Effects of stocking rate on SGR (g) – Mean initial weight (g) The Specific Growth Rate (SGR) of Rohu and Specific growth rate (SGR) and Feed Conversion Cauvery white carp across treatments is presented Ratio (FCR) were calculated at the end of the in Figure 1. The highest SGR of 0.59 was found in experiment for each treatment using formulae Rohu cultured in T2, followed by 0.58 in T3, 0.50 developed by Hopkins (year). in T4 and 0.44 in T1. Similarly, in Cauvery white LogW2 –LogW1 carp, the highest SGR of 0.59 was found in T1, SGR (% per day) = × 100 T followed by 0.52 each in T2 and T3 and lowest SGR of 0.48 in T4. SGRs recorded in his study Where, W2 = mean final weight (g), W1 = mean were comparable with the findings of a study of initial weight (g), T is culture period (days). Hossain et al. (2008) who reported similar SGR. In iii. Feed Conversion Ratio (FCR): this study, the high SGRs of Rohu and Cauvery white carp were in T2 and T1, respectively, which Total feed consumed (kg) FCR = could be due to low stocking rate as compared to Total weight gained (kg) T3 and T4.
2.6 Data analysis 3.3 Effects of stocking rate on FCR of Rohu and The dataset was analyzed using multivariate Cirrhinus mrigala ANOVA. Since the ponds were not replicated, Rohu exhibited best FCR value in T2 (0.97) and analysis was carried out separately for each fish lowest FCR value in T4 (1.08) (Figure 2). On the species. Stocking density was an independent other hand, Cauvery white carp had highest FCR in variable and fish body length and weight were T1 (0.98) and T2 (0.98) and lowest in T4 (1.04). dependent variables. The difference in the means of Similar results are reported by Manomaitis et al. dependent variables among stocking densities was (2004) and Islam et al. (2014). However, this result considered significant when p value was less than contradicts with the findings of Kausar and Salim 0.05. Statistical software SPSS version 23.0 was (2006) and Saeed et al. (2005) who reported high
Table 1: Water quality parameters in different treatment ponds during experiment. (ns-nonsignificant). -1 Treatment pH DO2 (mg L ) Temperature (°C) Transparency (cm) 2 fingerlings m-2 7.69 ± 0.43 5.41 ± 0.98 29.18 ± 2.04 27.57 ± 1.63 4 fingerlings m-2 7.74 ± 0.45 5.31 ± 1.16 29.34 ± 2.10 27.64 ± 1.72 6 fingerlings m-2 7.64 ± 0.39 5.27 ± 0.99 29.16 ± 2.12 27.64 ± 1.72 8 fingerlings m-2 7.65 ± 0.36 4.79 ± 1.09 29.32 ± 2.16 27.64 ± 1.72 p value ns ns ns ns
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Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 26-30, 2018
FCR values in Rohu. Cauvery white carp in Bangladesh. In this study, In this study, the best FCR of Rohu and the highest performance of fishes cultured in T2 Cauvery white carp in T2 and T1, respectively, could be due to low stocking rate and its could be due to low stocking rate and consequent consequent stocking densities that provides ideal low stocking densities. pond ecosystem.
3.4 Effects of stocking rate on growth 4. CONCLUSION performance of Rohu and Cauvery white carp The stocking rate of four fish m-2 exhibited the The fingerlings of Rohu in T2 had a significantly highest growth performance by both fish species. higher final growth performance, followed by T3, Therefore, the stocking rate of four fish m-2 is T and T4 (Table 2). Similarly, the growth 1 recommended for successful polyculture in areas performance was higher in T1 than T4. Similar that lie within the similar agro-ecological zones of growth performance result is reported by Mamun this study. and Mahmud (2014) and Basak et al. (2017) on
Rohu in Bangladesh.
0.8 2 fingerlings per square meter 4 fingerlings per square meter 6 fingerlings per square meter 8 fingerlings per square meter
0.6
0.4
0.2 SpecificGrowth Rate (%) 0 Labeo rohita Cirrhinus mrigala Figure 1: Specific Growth Rate (SGR) of Rohu and Cauvery white carp across treatments.
1.12 2 fingerlings per square meter 4 fingerlings per square meter 6 fingerlings per square meter 8 fingerlings per square meter 1.08 1.04 1.00 0.96
0.92 Feed Conversion Ratio 0.88 Labeo rohita Cirrhinus mrigala Figure 2: Feed Conversion Ratio (FCR) of Rohu and Cauvery white carp across treatments. Similarly, the highest growth performance by REFERENCES Cauvery white carp was recorded in T2. Unlike Rohu, Cauvery white carp cultured in T1 had the Basak SS, Rahman MM, Bashar MA, Khan MMH second highest growth, followed by T3 and T4. and Mahmud MY (2017). Production and Growth performance was significantly higher growth performance under carp polyculture in T2 than T1. T2 had a greater initial growth with different stocking densities in hilly creeks performance than T3. Although, statistically not of Kaptai Lake, Rangamati. MOJ Ecology and significant, the growth performance of T3 was Environmental Science, 2 (4): 1-5. greater than T4 (Table 2). Similar growth Das DR, Alam MN, Choudhury BBP, Faroque performance result is reported by Mamun and MAA and Haque MA (2011). Production Mahmud (2014) and Basak et al. (2017) on Thinley et al. (2018) 29
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Table 2: Initial and Final Body Weight (BW) & Body Length (BL) of fish species (Mean ± SD). Means with different letters within the column differs significantly at p≤0.05. Superscript with different letters within the column differs significantly at p≤0.05. Rohu Cauvery white carp (BW in g/BL in cm) (BW in g/BL in cm) Initial Growth Growth Body (g) (g) Weight (final- (final- Final Body Initial Body Final Body and body initial initial Weight and Weight and body Weight and length weight) weight) Treatment body length length body length
2 fingerlings m-2 8.02/8.79 138.5/22.5ac 130.48 7.50/9.30 153.1/24.2ac 145.5 4 fingerlings m-2 8.09/8.61 274.9/26.8b 266.79 7.47/9.11 171.6/24.8b 164.17 6 fingerlings m-2 8.14/8.79 141.7/21.7ad 133.52 7.53/9.19 133.8/22.7ad 126.22 8 fingerlings m-2 8.12/8.79 135.7/21.9cd 127.59 7.48/9.20 110.7/21.8cd 103.24 performance of different indigenous and exotic cirrhinus cirrhosus) and their hybrids. Journal carps at different densities in pond polyculture of Fisheries and Aquatic Science, 6: 1-17. system. Journal of Agroforestry and Manomaitis L, Cremer MC and Anand PEV Environment, 5 (1): 31-36. (2004). Growth performance of Rohu carp in Haque MR, Rahman MS, Islam MS, Ali MM, ponds using the ASA feed-based technology Islam MR and Shahabuddin AM (2015). with soy-maximized feed compared to the India Production and Growth Performances of Carps national package methodology. American in Different Stocking Densities of Polyculture. Soybean Association, St. Louis, USA. World Journal of Zoology, 10 (4): 247-251. Saeed M, Salim M and Noreen U (2005). Study on Hossain KZ, Miah MJU and Ali MH (2008). the growth performance and feed conversion Effects of inorganic fertilizers on the growth ratio of Rohu fed on soybean meal, blood meal and production performance of exotic carps in and corn gluten 60%. Pakistan Veterinary polyculture system. Progress Agriculture, 19 Journal, 25 (3): 121-126. (2): 129-137. Hossain MY (2000). Effects of iso-phosphorus organic and inorganic fertilizer on water quality parameters and biological production. Department of Fisheries management, Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh, Bangladesh. Islam M, Ahsan DA, Mandal SC and Hossain A (2014). Effects of salinity changes on growth performance and survival of Rohu fingerlings, Rohu (Hamilton 1822). Journal of Coastal Development, 17 (1): 1-6. Jena JK, Ayyappan S, Aravindakshan PK and Muduli HK (2001). Comparative evaluation of growth, survival and production of carp species at different stocking densities under polyculture. Indian Journal of Fisheries, 48 (1): 17-25. Kausar R and Salim M (2006). Effect of water temperature on the growth performance and feed conversion ratio of Labeo rohita. Pakistan Veterinary Journal, 26 (3): 105-108. Mamun AA and Mahmud AI (2014). Study on the growth performance and production of juvenile Indian Major Carps (Catla catla, Rohu and
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Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page: 31-36, 2018
Full length paper Exotic pigs receive different fattening periods from Bhutanese farmers
TENZIN PENJOR1*, GYEMBO TSHETEN1, PEMA SHERAB1 AND MIN P TIMSINA2
1National Piggery Research Centre, Department of Livestock, Ministry of Agriculture and Forests, Gelephu, Sarpang, Bhutan. 2Department of Livestock, Ministry of Agriculture and Forests, Thimphu, Bhutan.
*Author for correspondence: [email protected].
Copyright © 2018 Tenzin Penjor. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: The objectives of the study were to assess the fattening period followed by Bhutanese pig farmers for exotic breeds of pig and estimate the final carcass weight at village level. A close ended questionnaire was used for the field survey. In total, 274 households were interviewed between February to April, 2017. Respondents either owned pigs at the time of interview or had fresh experience of rearing pigs. About 77% of respondents owned exotic breeds of pigs and the remaining 23% reared local breeds of pigs, including the crosses. About 63% of respondents fattened exotic pigs for duration more than 11 months before slaughter, while about 37% of respondents fattened for more than 12 months. Over 68% of respondents achieved carcass weight of more than 70 kg per pig. Generally, about 42% of respondents achieved carcass weight of more than 70 kg per pig within the fattening duration of 8-11 months. About 82% of respondents practiced wet feeding comprising thin stillage (waste from a distillery of Army Welfare Project) and kitchen wastes mixed with other locally available feed resources. However, such feeding practices appear to have not met the nutritional requirement of pigs, which likely contributed to slow growth and prolonged fattening period. To achieve more carcass weight in short duration, farmers need to adopt proper feeding management with balanced ration.
Keywords: Carcass weight; exotic breed; farming; fattening duration; pig.
1. INTRODUCTION require much space; have prolific breeding potential; and are docile. These factors not only Livestock production is an important component of lead to increased profitability but also help in agricultural economy of developing countries meeting the growing demand for meat. These (Sugiyama et al. 2003). The contribution of attributes make pig farming a viable and profitable livestock to Gross Domestic Product is 24% enterprise that can be easily taken up by poor (MoAF 2013). Among livestock, pig production farmers. plays an important role in alleviating rural poverty. Pigs have ability to convert inedible food into In South East Asia, pig constitutes 48% of the total meat and are often fed with household food livestock population (FAO 2011). Pigs have fast waste when pigs are kept near homestead. Pig growth rates and good feed-to-meat conversion farming adds value to local food waste and crop ratios; are relatively easy to raise, and do not residue, which otherwise would be unsuitable for 31
Bhutan Journal of Animal Science (BJAS)Volume 2, Issue 1, Page 31-36, 2018 food production. In this way, pig production 2. MATERIALS AND METHODS contributes to sustainable local agriculture by local 2.1 Study areas mineral cycle and removing waste (Oosting et al. A survey was conducted in eight purposively 2014). selected districts covering 22 subdistricts (Figure Market weight is considered as an important 1). The selected districts were mostly from southern economic factor in pig farming as it influences Bhutan as pig farming in this region has minimal profit (Kim et al. 2005). Profitability of pig social stigma attached to the rearing and slaughter enterprise is judged mainly by the length of of pigs (Timsina and Sherpa 2005). fattening period. Generally, exotic pig breeds are known for better growth within a year. However, to 2.2 Data collection date, no attention was given to evaluate the exact A close-ended questionnaire was used to collect fattening duration for pigs under Bhutanese farming information from the respondents. The condition. Moreover, there are no established questionnaire was pre-tested with four pig farmers scientific evidences on the economical fattening in Sarpang district and changes were made where period of pigs in the country. One of the main necessary. The survey was conducted between targets of pig sector development in 11th plan was February to April, 2017. Face to face interview was to achieve carcass weight of about 80 kg within conducted with 274 respondents in the study areas. nine months of fattening duration. Although, exotic The questionnaire consisted of 15 closed ended pigs are reared by farmers, there is lack of scientific questions, excluding respondents’ details. The evidence on the actual fattening period followed by questionnaire broadly covered areas such as pig farmers. This information is important in fattening duration and carcass weight of exotic pig designing future interventions. Therefore, a breeds. Besides, the information on pig breeds, sex, questionnaire survey was conducted in the selected production system, feed types, farm size and animal pig farming areas. The study objective was to gain health services were also collected. better understanding on fattening duration followed by farmers for exotic pig breeds and estimate the 2.3 Data analysis final carcass weight under farmers’ management The survey data were entered in Microsoft Excel condition. sheet after coding questions and responses.
Figure 1: Study areas.
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Descriptive statistics was used to analyze the backyard farming. It is similar to Muys and dataset and the results were presented in Westenbrink (2004) who reported that small farms percentages. Graphs and tables were generated require minimum amount of inputs and lesser time using Microsoft Excel. Cross tabulation was carried and investment. But it must be noted that backyard out to determine carcass weight at different farms may not necessarily contribute to national fattening duration. The data were statistically pork self-sufficiency (Oosting et al. 2014), although analyzed using SPSS version 23. it enhances rural livelihood situation and availability of protein to improve household 3. RESULTS AND DISCUSSION nutrition.
3.1 Respondents and farming 3.3 Feeds and feeding The demographic profile of respondents is The backyard level pig farmers usually practiced presented in Figure 2. Forty-six percent of the dry and wet feeding system (Figure 4). About 82% respondents had completed primary or non-formal of the respondents practiced wet form of feeding, education, 15% with high secondary school and 1% using locally available feed resources, including of the respondents completed college education. thin stillage. The remaining 18% practiced dry However, 38% of the respondents were illiterate. form of feeding, using concentrates. The thin Pig farming in the sampled pockets was dominated stillage used in wet feeding is a waste from by integrated farming system. About 80% of the distillery of Army Welfare Project, which has total respondents were involved in rearing two or more solid of 2.5–3% with specific gravity of 1.002–
50 45 40 35 30 25 20 15
Respondents (%) 10 5 0 Primary/Non Formal Illeterate/uneducated Higher secondary College education Education school education
Figure 2: Demographic profile of respondents. types of farm animals such as pig with poultry, pig 100 with fish and pig with other domestic animals, besides agricultural cropping. On the contrary, 20% 90 of the respondents were found to rear only pigs. 80 70 3.2 Category of pig farms 60 Figure 3 presents the category of pig farms found in the study areas. Majority (86%) of respondents 50 practiced backyard pig farming with <10 pigs per 40 household and the pigs were raised mainly for meat Respondents (%) 30 purpose. About 9% of the pig farms were semi- commercial type with 10-49 pigs per household and 20 only 2% were commercial farms with 50-100 pigs 10 or more per household. By and large, the pig farms 0 were generally managed by family members regardless of farm size.
It was observed that the farm size of backyard
Semi- Backyard
piggery farm was about two pigs per farm. Majority (<10pigs)
(10-49 pigs)
commercial Commercial of respondents practiced backyard pig farming, (50-100 pigs) which could be due to less investment involved in Figure 3: Category of pig farms.
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1.004. Farmers practiced wet feeding, which was fattened exotic pigs for duration of less than 11 intended to meet optimal nutritional requirements months, while 37.5% of respondents fattened for of pig. However, through informal interviews, it more than 12 months. Regardless of fattening was noted that pig farmers appeared not to have duration, over 68% of respondents achieved carcass adequate technical knowledge on blending local weight of more than 70 kg for exotic pig. feed ingredients for feeding their animals. However, it was found that the remaining Missotten et al. (2015) reported that the ratio of raw respondents fattened exotic pigs for a year or more materials and water should ideally range from 1:1.5 to achieve the similar carcass weight. to 1:4. In order to reduce the cost of production and According to Huynh et al. (2007), farms in maximize profit, most of the respondents in the Cambodia with 2-4 pigs usually follow fattening sampled areas minimized production inputs through period of 8-12 months to achieve marketable feeding locally available feed resources. The weight. Although, there is no best weight to decide respondents are of the opinion that feeding at which pigs are to be marketed (Lawlor 2010; concentrate feeds incurs huge cost. It appears true Plain 2010), the profitability of fattening farm is since feed accounts for about 70% of the total determined by short fattening period (Stender 2012) production cost (Huynh et al. 2007). Under and higher market weight to certain point (Kim et backyard system, regardless of the situation, access al. 2005). In our context, owing to limited technical to quality and quantity of locally available feed knowledge of farmers, the pigs in the backyard resources determines the protein supply to growing system are usually reared and fattened for more pigs and their slaughter weight. Njoku et al. (2015) than a year, which may not be economical. It could reported that both quality and quantity of feed fed be due to poor and imbalanced nutrition leading to to the growing pigs have great influence on carcass prolonged fattening duration and low carcass weight. weight of exotic pig. According to Plain and Lawlor (2010), the slaughter weight is decided by many factors such as 90 age, space, weight, performance, genetics, buyers’ 80 preference, expected price change and convenience. 70 However, the target live weight of pigs at the time 60 of slaughter could be 110 kg for the most 50 economical strategy (Lawlor 2010). Most literatures show that pig slaughter weight differs 40 from country to country and from region to region.
30 Pig producers in European countries, including Respondents Respondents (%) 20 Denmark and Netherlands, market their pigs at 120- 10 130 kg (NASS 2003), whereas China and Korea market their pigs at 90-105 kg and 100-110 kg, 0 respectively (Kim et al. 2005). Relatively, slaughter Wet feeding Dry feeding
Figure 4: Type of feeding in pig farms. 90
3.4 Pig breeds 80 The pigs raised by the respondents in the sampled 70 pockets were both exotic and local breeds (Figure 5). About 77% of the respondents reared exotic pig 60 breeds and remaining 23% reared local pigs. Similar to the report of FAO (2011), our finding 50 shows that farmers prefer exotic pig breeds over 40 local pigs. It is probably because exotic breeds have higher carcass output and shorter fattening period. Respondents (%) 30 However, farmers with limited resources still prefer 20 to rear local pig breeds as they have better adaptability and can thrive well under adverse 10 climatic condition in low input production system. 3.5 Fattening duration and carcass weight 0 Exotic breed Local breed The fattening duration and the corresponding carcass weight of exotic pig breeds are presented in Figure 5: Exotic and local pig breeds reared by Table 1. Majority of the respondents (62.60%) farmers.
Penjor et al. (2018) 34
Bhutan Journal of Animal Science (BJAS)Volume 2, Issue 1, Page 31-36, 2018 weights in Asian countries are lower than Europe Acknowledgements and North America (Kim et al. 2005). In this study, The authors would like to immensely acknowledge the respondents expressed that they raised pigs until the logistic support of Extension Officials and local it weighed about 90-100 kg while some sold their leaders during the study. We thank Dr. Kesang pigs when they were in need of money. Similar Wangchuk, Principal Research Officer, Research finding was reported by Plain (2010). As it was and Extension Division, Department of Livestock, beyond the scope of the present study, we did not for his technical assistance in refining the make comparisons between local and exotic pig questionnaire and tutoring on SPSS. We are indeed breeds in terms of fattening duration and carcass grateful to Mr. Pema Thinley, Livestock Production weight under different feeding and management Officer, National Research Centre for Aquaculture condition. The need for such study in future is Gelephu for providing his assistance in data imperative to establish scientific evidences on the analysis. comparative advantages of raising different breeds of pigs under Bhutanese farming conditions. REFERENCES
FAO (2011). Molecular genetic characterization of Table 1: Carcass weight of exotic breeds of pigs at animal genetic resources. Health guidelines. different fattening period. Food and Agriculture Organization of United Fattening Carcass weight (kg) Nations. Rome, Italy. duration Gebremedhin MB (2016). Pig production under (month) 50-59 60-69 70-79 ≥ 80 Total small scale intensive farming in east Shewa of central Oromia, Ethiopia: Management, feed 8-9 4.7% 7.6% 9.0% 10.0% 31.3% resources, performance and marketing 10-11 1.9% 6.2% 13.3% 10.0% 31.3% practices. (PhD Thesis). Addis Ababa 12-13 1.9% 0.9% 5.7% 6.6% 15.2% University, College of Veterinary Medicine and >13 1.9% 6.2% 3.3% 10.9% 22.3% Agriculture, Ethopia. Total 10.4% 20.9% 31.3% 37.4% 100% Huynh TTT, Aarnink AJA, Drucker A and Verstegen MWA (2007). Pig production in Cambodia, Laos, Philippines and Vietnam: A 4. CONCLUSION Review. Asian Journal of Agriculture and Despite challenges confronted by farmers, the pig Development, 4 (1): 70-90. farming is still dominant in the study areas. It is Kim YS, Kim SW, Weaver MA and Lee CY evident that although the number of pig farmers (2005). Increasing the Pig Market Weight: over the years has declined, few of the sampled World Trends, Expected Consequences and households have ventured into semi-commercial Practical Considerations. Jinju, Korea: and commercial farming with 10-100 pigs. Besides, Regional Animal Industry Research Centre, quite a good number of respondents have more than Jinju National University. Asian-Australian three pigs in their backyard farms. Most pig Journal of Animal Sciences, 18 (4): 590-600. farmers fattened exotic pig breeds for less than a Lawlor P (2010). What is optimum slaughter year and achieved the carcass weight of more than weight for pigs? Pig Development Unit, 70 kg. The pig production cost was minimized by Animal and Grassland Research Centre, locally available feed resources. Such feeding Teagasc, Moorepark, Fermoy Co. Cork. practice did not help to achieve desirable market Available at: weight within 9-12 months of fattening period, https://www.teagasc.ie/media/website/publicati therefore, it is not economical. To encourage and ons/2010/1951/Optimum_slaughter_weights_ promote pig farming, government must provide May2010.pdf . (Accessed on July 7, 2017). enabling policy support and come up with strategic Missotten JAM, Michiels J, Degroote J and de interventions with better innovations. Focus should Smet S (2015). Fermented liquid feed for pigs: be given to commercialize pig production in the an ancient technique for the future. Journal of potential pockets where there is no social stigma. Animal Science and Biotechnology, 6: 1-9. There is also a need to develop skills and technical MOAF (2013). Agricultural Marketing Policy of capacity of farmers on improved pig feeding and Bhutan 2013. Ministry of Agriculture and management. There is a need for more scientific Forest. Thimphu, Bhutan. research to determine the economical fattening Muys D and Westenbrink G (2004). Keeping pigs th period of different pig breeds under Bhutanese in the Tropics. 4 Edition. Agromisa farming conditions. Foundation, Wageningen. Available at:
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http://www.journeytoforever.org/farm_library/ Plain R and Mintert J (2010). Marketing Slaughter AD1.pdf. Hogs: Where, How & When. Available at: (Accessed on July 7, 2017). http://articles.extension.org/pages/27212/marke NASS (2003). Agricultural Statistics. USDA. ting-slaughter-hogs:-where-when-how National Agricultural Statistics Service. US (Accessed on May, 17, 2017). Government Printing Office, Washington, D.C. Stender DR (2012). Swine feed Njoku CP, Adeyemi OA, Sogunle OM and Aina Efficiency:Influence of Market Weight. Iowa: ABJ (2015). Growth Performance, Carcass Iowa State University. Yield and Organ Weight of Growing Pigs and Sugiyama M, Iddamalgoda A, Oguri K, and Fed Different Levels of Feed. Slovak Journal of Kamiya N (2003). Development of livestock Animal Science, 48: 16-22. sector in Asia: An analysis of present situation Oosting SJ, Udo HMJ and Viets TC (2014). of livestock sector and its importance for future Development of livestock production in the development. Gifu University, Yanagido, tropics: farm and farmers’ perspectives. Animal Japan. Production Systems Group, Wageningen Timsina MP and Sherpa DL (2005). University, Wageningen, The Netherlands. Characterization of Bhutan’s indigenous pig Phengsavanh P, Ogle B, Stür W, Frankow- genetic resources and production systems. Lindberg BE, Lindberg JE (2010). Feeding and Bajo, Bhutan, Renewable Natural Resources performance of pigs in smallholder production Research Centre, Council of RNR Research of systems in Northern Lao PDR. Tropical animal Bhutan, Ministry of Agriculture. health and production 42: 1627–33.
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Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 37-41, 2018
Full length paper Production performances of crossbred pigs in government farms
LOKEY THAPA1* AND MIN P TIMSINA2
1National Dairy Research Centre, Department of Livestock, Ministry of Agriculture and Forests, Yusipang, Thimphu, Bhutan. 2Department of Livestock, Ministry of Agriculture and Forests, Thimphu, Bhutan.
*Author for correspondence: [email protected].
Copyright © 2018 Lokay Thapa. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: This study was carried out with the objective to evaluate and compare production performances of crossbred pigs between two government multiplier farms at Lingmethang and Serbithang, falling under two different regions. A total of 735 numbers of farrowing data were analyzed from the Elite herd database, maintained by these farms for a period of 5 years from July 2006 to June 2011. The pig breeds under study were Large Black (LB), Saddleback (SB) and Hampshire (H). The study parameters were litter weight at birth, piglet weight at weaning, age at first service, weaning to service interval, farrowing interval, litter sizes at birth and litter size at weaning. Two-sample t-test was conducted. The mean litter weights at birth in LB, SB and H crossbred pigs were greater in Lingmethang farm. The weaning weight, mean weaning age, mean age at first service, mean litter size at birth, and litter size at weaning of all three breeds were also greater in Lingmethang farm. Lingmethang farm had a shorter mean weaning to service interval for all breeds. The study concluded that reproduction and production parameters of crossbred pigs raised at Lingmethang farm are better than those raised at Serbithang farm. Based on performance record, the SB crossbred pigs could be a better breed for warm places. Similarly, LB and H performed better in cold places and could be suitable for temperate environment.
Keywords: Hampshire; Large black; litter; pig; Saddleback; weaning.
1. INTRODUCTION population in the country (DoL 2016). Crossbreeding of indigenous pigs with exotic or Livestock is one of the important components of improved breeds was initiated by the Government farming system in Bhutan. About 10% of rural of Bhutan since early 1960s with the main aim to households rear pigs (DoL 2016) Bhutanese people enhance and improve income and dietary protein of raise pigs for meat production and to generate cash rural people (Nidup et al. 2011). income. However, due to religious sentiments, pig Currently, there are three government pig farming is still at the subsistence level, generating breeding farms in Bhutan; National Pig Breeding small supplementary income for the farmers. Most Centre at Serbithang in Western Bhutan, Regional pigs raised in rural areas are indigenous breeds, Pig and Poultry Breeding Centre at Lingmethang in constituting more than 60% of the total pig 37
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 37-41, 2018
Eastern Bhutan, and National Pig Research Centre service, number of services per conception, litter (NPiRC) at Gelephu in South-Central Bhutan. size at birth, litter weight at birth, piglet weight at NPiRC is the nucleus farm and farms at Serbithang weaning, age at weaning, pre-weaning mortality, and Lingmethang are multiplier farms. weight at weaning and weaning to oestrous interval. The progenies produced by the nucleus farm are A total of 735 production and reproduction related distributed mainly to the multiplier farms for further information was collected to evaluate the breeding and multiplication. The multiplier farms production efficiency of the farm. raise Saddleback (SB) cross, Large Black (LB) cross, Duroc, and Hampshire (H) breeds of pigs for 2.3. Data analysis further multiplication. For the last four decades, the The dataset was entered in Microsoft Excel and multiplier farms were mandated to carry out exported to SPSS. Two-sample t-test was crossbreeding to produce hybridized piglets for performed to compare production parameters further distribution to needy farmers in all potential between two multiplier farms. Multiplier farms pig rearing areas across the country. Although, for were independent variables and production many years, piglets were distributed by nucleus parameters were dependent variables. Differences farm to multiplier farms, no assessments have been between parameters were considered significant carried out to date to understand the production when p values were less than 0.05. The entire performances of pigs in the multiplier farms. A dataset was analyzed with SPSS version 16.0 (SPSS proper understanding of production performance is Inc. Chicago, Illinois, USA). important as it will provide a basis for setting bench mark for these farms to improve production 3. RESULTS AND DISCUSSION efficiency. Therefore, a study was conducted with 3.1 Litter weight at birth and piglet weight at the objective to evaluate and compare production weaning performances of pigs between two government The mean litter weight at birth and weaning weight multiplier farms under two different regions. of piglets at Lingmethang and Serbithang are
presented in Table 1. The mean litter weights at 2. MATERIALS AND METHODS birth in LB, SB and H crossbred pigs were greater 2.1 Pig breed in Lingmethang farm. This is probably due to warm The production data of 61 sows at Lingmethang and condition at Lingmethang and litter weight at birth 90 sows at Serbithang, were compiled from Elite has been reported to be heavier in hot places herd database maintained by these two farms. The (Jourdine et al. 2006; Belstra et al. 2004). In this sows at Lingmethang were progenies of LB boar study, pigs achieved the highest litter weight at crossed with SB and H. The sows at Serbithang birth from 3rd to 7th parity, which agrees with the were progenies of LB and H, sired by SB breed. In findings of Roongsitthichai et al. (2010) and Yuzo this study, the information was collected from those et al. (2017). sows, which had all the performance data records Similarly, the weaning weight of all three available till 8th parity. breeds was higher in Lingmethang farm. Among breeds, the weaning weight of SB crossbred piglets 2.2. Data collection was found to be comparatively higher than other The information was collected on sow breeds, which could be due to good sow mother and identification, date of birth, breed, age at first excellent milker, besides having heavier litter Table 1: Birth weight and weaning weight of pig breeds in two government farms. Breed Farm Birth weight (kg) Weaning weight (kg) Weaning Age (days) LB×LB Lingmethang 9.88 69.95 48.20 Serbithang 8.68 64.81 39.63 p value *** ns *** SB×SB Lingmethang 10.67 83.14 46.40 Serbithang 8.60 64.81 38.37 p value *** *** *** H×H Lingmethang 10.25 77.72 48.04 Serbithang 8.92 60.59 39.09 p value ** *** *** **p≤0.01, ***p≤0.001, ns-nonsignificant
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Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 37-41, 2018 weight at weaning. weaning to service interval results in less number of The mean weaning age was recorded higher in nonproductive days and high number of pigs Lingmethang farm. Among breeds, the weaning age farrowed or weaned. was greater for LB crossbred pigs. The weaning age The mean weaning to service interval was of piglets in different farms was determined by the shorter for all breeds at Lingmethang. Among bench mark for piglet’s wean weight set by breeds at Lingmethang, the weaning to service Department of Livestock. Generally, Serbithang interval was shortest for H and longest for LB. The farm weans piglets when they are about 8 kg, interval was 40% better at Lingmethang than whereas Lingmethang weans piglets when they are Serbithang farm. This could be due to warm about 10 kg. These practices are similar to those climatic condition at Lingmethang since other reported for SB crossbred pigs (Gatenby and management factors such as feeding and housing Chemjong 1992). Increasing weaning age were similar. Weaning to service interval was about improves wean-to-finish growth performance, 30 days in gilts in both farms and was found to be growth and reduces mortality rate (Main et al. very long, which ideally happens in first and second 2004). parity sows. Although, 82% of services were done within 10 days after farrowing, the conception rate 3.2 Age at first service, weaning to service was found to be low in both farms, resulting in interval and farrowing interval repeated heat in sow. Seventy percent of breeding Age at first service, weaning to service interval, and occurred within 6 days of post weaning, which is in farrowing interval are presented in Table 2. The agreement with the findings of Wilson and Dewey mean ages at first service at Lingmethang and (1993). Weaning to service interval of lactating sow Serbithang were 550 and 418 days, respectively. In becomes longer when a sow loses its body weight general, the mean age at first service was recorded by more than 15% (Kunavongkrit and Heard 2000). highest for SB crossbred pigs at Lingmethang farm. There was a significant difference in age at first 3.3 Litter sizes at birth and weaning service for SB crossbred pigs reared in two farms The mean litter size at birth in Lingmethang and and could be due to its location at different climatic Serbithang are presented in Table 3. The mean litter zones. LB crossbred pig reached age at first service size at birth was highest in Lingmethang. Among earlier at Serbithang, which could be due to LB breeds, the litter size at birth was greater for SB being a breed suited for temperate environment. crossbred pigs. Kumaresan et al. (2007) also However, the age at first service at two farms is reported greater litter size at birth for SB crossbred higher compared with other countries. Ideally, the pigs. This study indicated that litter size at birth age at first service in any of the pig farms should be increases with increase in parity number, reaching a between 8 and 9 months and if it crosses more than plateau and then declines from 7th parity onwards. 10 months, then it is advisable to cull these animals This finding agrees with that of Peadar et al. (Rymer and Grant 2009). (2007). The gradual decline in litter size at birth Weaning to service interval is defined as the from 7th parity onwards could be due to age at first number of days from weaning to the day the female service, body weight, ovulation rate and is bred again. It is important since the shorter implantation rate. Thapa (2012) reported lower
Table 2: Age at first service, weaning to service interval and farrowing interval of big breeds in two government farms. (ns-nonsignificant). Breed Farm Age at First Weaning to service Farrowing interval (days) service (days) interval (days) LB Lingmethang 468 20.07 182.81 Serbithang 423 33.34 188.66 p value ns ns ns
SB Lingmethang 588 19.92 181.53 Serbithang 439 26.87 179.37 p value *** ns ns
H Lingmethang 473 11.77 174 Serbithang 523 24.4 178.18 p value ns ns ns
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Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 37-41, 2018 litter size at birth in pure pig breed when compared index, farrowing rate and reduce weaning age in with crossbred pigs. Young et al. (1976) also piglet with efficient management of lactating and reported greater birth weight of crossbred pigs due dry sows. to hybrid vigor than pure breeds. The number of pigs weaned depends on the REFERENCES number of piglets born alive and preweaning Kunavongkrit A and TW Heard (2000). Pig mortality. In this study, the litter size at weaning reproduction in South East Asia. Animal was greater for Lingmethang farm. Among breeds, Reproduction Science, 60–61: 527–533 the litter size at weaning in SB crossbred pigs was Alison L, Smith SK, Serenius TV, Baas TJ and greater, followed by LB crossbred pigs. This WMJ (2007). Effect of weaning age on nursery indicates that SB crossbred pigs can perform better pig and sow reproductive performance. Journal in any climatic condition, irrespective of of Swine Health and Production, 16 (3): 7. geographic locations. The SB breeds have better Andersen Inger Lise, Synne B and Egil BK (2005). mothering abilities, which is indicated by more Crushing of piglets by the mother sow -purely number of piglets weaned than other breeds. accidental or a poor mother. Applied Animal Similar finding was reported by Eugene et al. Behaviour Science, 93: 14. (1999) that litter size at weaning differs amongst Belstra B (2003). Parity associated changes in different crossbred pigs under different climatic reproductive performance: Physiological basis condition. on record keeping artifact. North Carolina State University. Table 3: Litter sizes at birth and weaning of pig Belstra BA, Flowers WL and See MT (2004). breeds in two government farms. Factors affecting temporal relationships Breed Farm Litter size Litter Size at between estrus and ovulation in commercial at birth weaning (no.) sow farms. Animal Reproduction Science, 84: (no.) 377-394. DoL (2016). Livestock Statistics. Department of LB Lingmethang 8.36 7.97 Livestock, Ministry of Agriculture and Forests, Serbithang 7.99 7.27 Thimphu, Bhutan. p value ns *** Eugene BL, Jenny-Ann LML, Simon JM, Alberto AT and Cotiw BS (1999). The reproductive SB Lingmethang 8.72 8.36 performance of sows raised by smallholder Serbithang 8.04 7.43 farmers in the Philippines. Preventive p value *** *** Veterinary Medicine, 41: 16. Filiz Akdag, Arslan S and Demir H (2009). The H Lingmethang 8.31 7.56 Effect of Parity and Litter Size on Birth Weight Serbithang 8.28 7.77 Variation on the Weaning Weight and Pre- p value ns ns Weaning Survival in Piglet. Journal of Animal ***p≤0.001, ns-nonsignificant and Veterinary Advances, 8(11): 6. Gatenby RM and Chemjong PB (1992). Reproduction of pigs in the hills of Eastern 4. CONCLUSIONS Nepal. Tropical Animal Health and Production, The reproduction and production parameters of 24: 8. crossbred pigs are better at Lingmethang farm than Kumaresan A, Bujarbaruah KM, Pathak KA, those raised at Serbithang farm. Based on Chhetri DSK and KAS (2007). Performance of performance record, the SB crossbred pigs could be pigs reared under traditional tribal low input a better breed for warmer places. Similarly, LB and production system and chemical composition of H performed better in colder places and could be non-conventional tropical plants used as pig suitable for temperate environment. From this feed. Livestock Science, 107: 5. study, it is also evident that litter size at birth Lay DC, Matteri RL, Carroll JA and Fangman TJ increased in both farms from 3rd to 7th parity. (2002). Preweaning survival in swine. Journal Thus, measures should be taken by the farm of Animal Science, 80: 13. management to retain high number of sows Alonso-Spilsbury M, Ramirez-Necoechea R, between these parities in order to maximize litter Gonzlez-Lozano M, Mota-Rojas D and. size and improve farm production efficiency. To Trujillo-Ortega ME (2007). Piglet Survival in minimize long farrowing interval, the farms have to Early Lactation: A Review. Journal of Animal improve weaning to service interval, farrowing and Veterinary Advances, 6: 76-86. 40
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Peadar GL and Brendan PL (2007). A review of Wilson MR and Dewey CE (1993). Association factors influencing litter size in Irish sows. Irish between weaning to estrus interval and sow Veterinary Journal, 60: 359-366. efficiency. Swine Health and Production, 1(4), Main RG, Dritz SS, Tokach MD, Goodband RD 6. and Nelssen JL (2004). Increasing weaning age Young LD, Johnson RK &and ITO (1976). improves pig performance in a multisite Reproductive performance of swine breeds to production system. Journal of Animal Science, produce pure bred and two cross bred litters. 82:1499–1507. Journal of Animal Science, 74: 16. Nidup K, Tshering D, Wangdi S, Gyeltshen C, Koketsu Y, Tani S and Iida R (2017). Factors for Phuntsho T and Moran C (2011). Farming and improving reproductive performance of sows biodiversity of pigs in Bhutan. Animal Genetic and herd productivity in commercial breeding Resources, 48: 15. herds. Porcine Health Management, 3:1. Rymer TS and Grant W (2009). Latest international developments in genetics and Research & Development from the JSR technical team. JSR Genetics Limited. Rafael AC, Xi L, Joy MC, Adam JM and Jack O (2012). Influence of birth order, birth weight, colostrums and serum immunoglobulin G on neonatal piglet survival. Journal of Animal Science and Biotechnology, 3: 42. Whiting TL and Plasma T (2008). Isolated weaning Technology: Humane benefits and concern in the productiob of pork. Canadian Veterinary Journal, 49(3): 9.
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Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 42-48, 2018
Full length paper Effects of weaning age on growth, health and mortality of piglets in government pig farm
SINGAY OM1*, PEMA SHERUB1 TSHERING GYELTSHEN2 AND MIN P TIMSINA3
1National Piggery Research and Development Centre, Department of Livestock, Ministry of Agriculture and Forests, Gelephu, Sarpang, Bhutan. 2College of Natural Resource, Royal University of Bhutan, Lobesa, Punakha, Bhutan. 3Department of Livestock, Ministry of Agriculture and Forests, Thimphu, Bhutan.
*Author for correspondence: [email protected].
Copyright © 2018 Singay Om. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: A study was conducted with the objective to evaluate the effects of weaning age on growth, health and mortality of piglets in a government farm. Forty-two piglets belonging to breed Duroc, Saddleback and Large Black, were selected randomly from piglets of five sows and were assigned to three treatments. The treatments were; T1 (28 days weaning age), T2 (35 days weaning age) and T3 (42 days weaning age). Each treatment was allotted 14 piglets. Piglets were weighed on day zero, weekly and on the day of weaning till 60 days. Daily post weaning feed consumption for each group was recorded. Incidences of diseases and mortality in all groups were recorded in pre-weaning (0 to 28 days) and post-weaning (28 to 60 days) periods. The mean final weight gain at 60 days was highest in T3, followed by T2 and T1. However, there was no significant difference among treatments in weight gain. Also, the survival in pre-weaning was similar in all treatments. Post-weaning survival was highest in T3 and lowest in T1, although there was no significant difference in survival among groups. The findings suggest that piglets can be weaned at an earlier age than the currently followed age (48 days) in the government farm, provided there is proper management and improved facilities in farrowing and nursery sheds.
Keywords: Age; body weight; mortality; piglet; survival; weaning.
1. INTRODUCTION (2015), the import of pork in 2014 was 2165 Metric Tons (MT) against domestic pork production of only Pigs play an important role in sustaining livelihoods 462.46 MT. One of the contributing factors to these of rural poor and pigs are seen as an entry point for existing issues could be due to large weaning ages in poverty alleviation (Biryomumaisho and Ogala the government pig farms. Thapa (2011) reported 2007). Fulfilling piglet demands of farmers has 48.78 days as weaning age in NPiRDC, Gelephu and always been a challenge for the government farms. 46.77 days in RPBC, Lingmethang. Sow loses more In 2013, the piglets demand from 18 districts was than 15% of body weight during long lactation and 9,000 and the government farms could supply only results in longer unproductive days (Quensel et al. 4,500 piglets (Dema 2016). The demand for pork is 2008). Late lactation decreases the number of piglets met through import from neighboring countries like produced per year as sows do not come into heat India and Nepal (DoL 2007). According to MoAF 42
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 42-48, 2018 when it has a nursing litter while early weaning age 2.4 Data analysis increases piglets per sow per year. However, the Average Daily Feed Intake (ADFI) and Average reported disadvantages of early weaning are reduced Daily Gain (ADG) were calculated in Microsoft growth rate, poor health and high mortality in piglets. Excel. Average Daily Feed Intake (ADFI) was These information shows that there is a need to calculated by dividing total feed consumed post identify an appropriate weaning age under weaning (for each weaning age) by the numbers of conditions found in government pig farms. pigs fed daily. Average Daily Gain (ADG) was Therefore, a study was conducted with the primary calculated by dividing body weight gain (Final objective to compare body weight gain and survival weight gain - initial weight) by feeding days. To of piglets under three early weaning ages till 60 days compare means of final weight gain and to determine of age. the effect of weaning, the analysis of covariance (ANCOVA) was conducted. Piglet survival were 2. MATERIALS AND METHOD analyzed using Kaplan-Meier survival model. Event in this study was the death or diseases occurred 2.1 Study area, treatments and sample size during the study period. Survival time was expressed The study was carried out at a pig farm of NPiRDC, in days. The pre-weaning survival was from 0 to 28 Gelephu, Sarpang district. The study was carried out days and the post weaning survival was after 28 to from December 2016 to February 2017. Mixed breed 60 days. of Duroc, Saddleback and Large Black were used.
There were three weaning treatments. The first 3. RESULTS AND DISCUSSION treatment (T1) was 28 days weaning age, second treatment 35 days weaning age and the third 3.1 Growth of piglets treatment 42 days weaning age. Piglets were selected The average body weights of piglets in three groups randomly with draw-lot method and assigned to were compared from 0 to 60 days of age (Figure 1). three treatment groups. Each treatment group had 14 All piglets in three treatments had similar average piglets. The sample size of 14 piglets was determined initial weights at the time of farrowing; 1.66 kg, 1.67 푠 sing the formula: n = 1 + 2c ( )² (Shah 2011). kg and 1.59 kg for T1, T2 and T3, respectively. The 푑 average weights of piglets in all treatments were also 2.2 Animal management similar until four weeks of age; 5.75 kg, 5.70 kg, 5.61 kg for T1, T2 and T3, respectively. However, in T3, Piglets were weighed individually using digital weighing balance and their ears were notched for the body weight of piglets increased after four weeks identification on the day of farrowing. Piglets were till six weeks of age (5.65 kg to 8.52 kg). After six weaned at 28, 35 and 42 days old and transferred to weeks (age at weaning), the weight increased slightly weaner shed. Each treatment group was maintained till seven weeks of age. In T1, the weight gained by in a pen measuring 3.14 m × 3.28 m. The pen had piglets was lower than T2 and T3 after four weeks complete solid concrete floors equipped with feeding (age at weaning). and drinking troughs. After seven weeks, piglets in all treatments Weaner pigs were fed starter feed mixed with showed increase in body weight. However, the final effective microorganism (EM) solution. Feed and weight gain at nine weeks (60 days) was slightly water were provided ad libitum throughout the higher in T3, although the difference was not experimental period. In-house temperatures were significant among groups. Gonzalez et al. (2015) and recorded in the morning, afternoon and evening. Cabrera et al. (2014) reported that late weaning gives better results than early weaning and the final weight 2.3 Data collection gains for late weaning are significantly greater than Individual piglets were weighed using a digital the early weaning. The average daily weight gain of weighing balance before feeding. Piglets in all piglets was found better under long nursing period groups were weighed on the day of weaning and (Gonzalez et al. 2015; Cabrera et al. 2014). This weekly thereafter until piglets were 60 days of age. could be due to minimal weaning stress experienced Feed consumption was recorded daily after weaning by the bigger and older pigs at weaning. Meulen et i.e. from 28 days, 35 days and 42 days old until 60 al. (2010) compared the stress hormonal level days old. Feed was weighed on daily basis before between late weaning at 7 weeks and early weaning feeding, using a digital weighing balance. Leftover at 4 weeks in Landrace and Large White pigs. The feed was collected and weighed daily and recorded. authors found that the level of stress hormone The incidences of mortality were recorded before cortisol was higher in piglets weaned at 4 weeks than weaning from 0 to 28 days and after weaning from those weaned at 7 weeks. 28 to 60 days in all three groups. 3.2 Effect of initial weight and weaning age on growth rate of piglets Om et al. (2018) 43
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 42-48, 2018
the low body weight at weaning and may have 12 28 days (Group I) affected the final weight gain. 35 days (Group II) 10 Table 1: Post-weaning ADG and ADFI under 42 days (Group III) different treatments 8 Parameters Weaning age
28 days 35 days 42 days 6
Body weight (kg) weight Body ADG (kg) 0.14 0.15 0.16 ADFI (kg) 0.27 0.38 0.43 4
3.3 Feed intake and weight gain 2 The piglets in T3 had higher Average Daily Gain (ADG) of 0.16 kg and piglets in T1 had lower ADG 0 of 0.14 kg (Table 2). Similarly, the piglets in T3 had 0 1 2 3 4 5 6 7 8 higher Average Daily Feed Intake (ADFI) compared to piglets in T1. This could be due to lower body Age (week) weight at 28 days weaning age and lower body weight at 60 days, compared to 35 and 42 days Figure 1: Weekly body weight of piglets weaning age. Our finding agrees with that of Colson weaned at different age. et al. (2005) and Wolter and Ellis (2001) who reported decrease in ADG of piglets weaned at 21 Table 1 presents the initial and final weights of and 28 days. piglets in three treatment groups. The mean initial In this study, though there was no reduction in body weights of piglets were 1.65±0.29 kg, body weight in T1 from the day of weaning (Figure 1.67±0.14 kg and 1.58±0.33 kg for group one, two 2), the ADG was lower compared to T2 and T3. This and three, respectively. The mean final body weight could be because of low body weight at weaning, at 60 days was 10.55±1.37 kg, 10.94±1.40 kg and therefore, ADFI of T1 was also lower than T2 and 11.13±2.36 kg for T1, T2 and T3, respectively. T3. Montsho et al. (2001) compared the late weaning However, the nonsignificant difference among at 35 days age with the early weaning at 21 days age treatments suggests that weaning age has no and found that piglets weaned at 21 days of age significant effect on final weight gain. This could be consumed more feed than those weaned at 35 days explained by reduced weaning stress due to good of age. This is because the early weaned piglets are facilities such as brooder for piglets, tarpaulin to more adapted to solid feed than late weaned piglets protect from cold wind, dry grass, concentrate feed (Montsho et al. 2001). Whereas, in this study, the and good welfare in the farm. Montsho et al. (2016) piglets when kept with sow did not consume much and Liliveld et al. (2013) reported that weaning has of solid feed. There are two possible reasons. Firstly, no effect on performance of piglets. However, in the sows provided enough milk that piglets did not their studies, the weaning ages were 21, 28 and 35 consume solid feed. Secondly, the low temperature days in Large White and Landrace cross in Ireland in nursery shed may have discouraged piglets from and Botswana. visiting the feeding tough. The mean final body weight was highest in T3 Table 2: Initial and final body weight of piglets (11.13±2.36 kg), followed by T2 (10.94±1.40 kg) under different treatments. Figures inside cells and T1 (10.55±1.37 kg). Similar findings were represent means±se. Means with similar letters are reported for Large White Yorkshire pigs weaned at not significantly different. 28, 42 and 56 days, where piglets weaned at older age were heavier than those weaned at younger age Weaning age (Jayashree and Sivakumar 2013; Vega et al. 2012; Parameter 28 days 35 days 42 days Wolter and Ellis 2001). This could be due to body weight gain at weaning. The lower body weight at Initial 1.65±0.3a 1.67±0.1a 1.58±0.3a weaning could be due to environmental stress (cold weight stress) as nursery shed in NPiRDC lacks brooding (kg) facilities and the in-house temperature ranges from 17˚C to 27˚C in December. The 14 to 28 days old Weight at 10.55±1.4a 10.94±1.4a 11.13±2.4a piglets require temperature range from 22˚C to 23˚C 60 days (Yan and Yamamoto 2000), which likely explains (kg)
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Figure 2: Survival of pre-weaning in three weaning groups.
Figure 3: Survival curve of post-weaning in three groups
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3.4 Pre-weaning mortality pathogens causing diarrhoea (Dong and Pluske None of the piglets died during the experimental 2007; Spreeuwenberg et al. 2004). period. The mean survival time at pre-weaning was Due to mixing of unfamiliar littermates, the similar across all three treatments but the mean piglet fighting was observed more in T1 and T2. survival time at post-weaning was highest in T3 and Piglets mixed at earlier age were reported to show lowest in T1 (Table 3). However, there was no aggression and fighting as pigs seek dominance significant difference among three treatments. The hierarchies (Colson et al. 2005; Boe 1991). This similar pre-weaning survival time in three groups could have affected the limbs of the piglets, resulting could be because there was equal number of piglets in swollen limbs. suffering from diarrhoea due to algalactic sow. The However, piglets in all treatments revived within piglets in all treatments were from same sow that a week and survived till 60 days and there was no suffered from algalactia and diarrhoea. The post-weaning mortality. This could be due to incidences of an event in pre-weaning from 0 to 28 improved brooding facilities such as electric heater, days were mostly around 6 days, however, there was dry grass bedding, tarpaulin curtains to protect from no pre-weaning mortality (Figure 3). The incidences cold wind, which maintain optimum temperature of diarrhoea in NPiRDC, Gelephu within first week required by the piglets. High post-weaning survival of age were also reported by Sherab (2012). in government piggery farms has been reported (Sherab 2012; Thapa 2011). Table 3: Mean (±se) survival time of pre-weaning and post-weaning of piglets. Means within the row 4. CONCLUSION with same letters do not differ significantly. Examining the weight gained at 60 days and with
Mean survival time (days) 100% post weaning survival, piglets can be weaned Weaning 28 days 35 days 42 days at an earlier age than the current practice of weaning at 48 days. However, the low weight of piglets Pre-weaning 23.6±2 a 23.3±2 a 23.4±2 a weaned at 28 days suggests that weaning at a very Post-weaning 47.2±4 a 49.2±3 a 56.7±2 a early age is not advisable. Proper farm management practices and improvement of facilities in the farrowing and nursery sheds are important to 3.5 Post-weaning mortality improve the body weight at weaning. Further, the The post-weaning mortality of piglets is presented in maintenance of mating at 0% inbreeding level would Figure 4. The incidences of diseases occurred mostly have low impact on growth and may improve pig around 28, 35 and 44 days. The event in T1 and T2 performance. occurred immediately after weaning, while it occurred later in T3. This could be because the Acknowledgements younger and smaller piglets exhibited more stress The authors would like to express profound gratitude than older and bigger piglets at weaning. Piglets to Dr. Tshering Gyeltshen, Lecturer, College of weaned at young age are reported to have high level Natural Resources (CNR) for guidance and of cortisol, indicating high stress (Meulen et al. 2010; assistance. Sincere gratitude to Mr. Pema Sherab, Worobec et al. 1999; Dividich and Herpin 1994). Program Director and all staff and attendants of The symptoms of diseased piglets were off feed, National Piggery Research and Development Centre, diarrhoea and swollen limbs, which occurred mostly Gelephu. In particular, the supports from Passang in T1, followed by T2 and T3. The stress arises Dema, Choiten Tenzin, Doku Tshering, and Tenzin mainly from maternal separation, mixing with Penjor, during the entire experimental period, are unfamiliar littermates, change in environment highly appreciated. (nursery to weaner shed) and change in diet (sow milk). Weaning stress compromises intestinal barrier REFERENCES function (Bruininx et al. 2001; McCracken et al. 1999). Masri et al. (2014) evaluated the small Biryomumaisho S Ogala R (2007). A study of pre- intestine of Landrace, Large White and their hybrids weaning piglet moratality in selected farms in and reported that weaning at 28 days caused dramatic the peri-urban areas of Kampala, Uganda. Africa structural changes in the mucosa than weaning at 35 Journal of Animal and Biomedical Science, 2 days and later. This results in diarrhoea and poor (1): 1-9. performance of newly weaned piglets (Pluske 2016; Bruininx EMAM, VanDerPeetSchwering CMC, Campbell et al. 2013). The low feed intake in the Schrama JW, Vereijken PFG, Vesseur PC, immediate post weaning period contributes to Everts H, Hartog LA and Beynen AC (2001). morphological changes in intestine, exposing it to Individually measured feed intake characteristics and growth performance of
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group- housed weanling pigs: Effect of sex, 233. initial body weight and body weight distribution http://dx.doi.org/10.1016/j.livsci.2013.06.030. within groups. Journal Animal Science, 79: 301- Masri SA, Hunigen H, Aiyan AA, Rieger J, Zentek 308. J, Richardson K and Plendl J (2014). Influence Boe K (1991). The process of weaning in pigs : of age at weaning and feeding regimes on the When the sow decides. Applied Animal postnatal morphology of the porcine small Behavior Science, 30: 47–59. intestine. Journal of Swine Health and Cabrera RA, Boyd RD, Jungst SB, Wilson ER, Production, 23 (4): 186-203. Johnston ME, Vignes, JL and Odle J (2014). McCracken BA, Spurlock ME, Roos MA, Impact of lactation length and piglet weaning Zuckermann FA and Gaskins HR (1999). weight on long-term growth and viability of Weaning anorexia may contribute to local progeny. Journal of Animal Science, 88: 2265- inflammation in the piglet small intestine, 2276. doi:10.2527/jas.2009-2121. Journal of Nutrition, 129: 613–619. Campbell JM, Crenshaw JD and Polo J (2013). The Meulen JVD, Koopmans SJ, Dekker RA and biological stress of early weaned piglets, Journal Hoogendoorn A (2010). Increasing weaning age of Animal Science and Biotechnology, 4(19): 2- of piglets from 4 to 7 weeks reduces stress, 4. increases post-weaning feed intake but does not Colson V, Orgeur P, Foury A and Mormede P improve intestinal functionality. Animal, 4 (10): (2005). Consequences of weaning piglets at 21 1653-1661. doi:10.1017/S1751731110001011. days and 28 days on growth, behaviour and Montsho TM, Moreki JC, Tsopito CM and Nsoso SJ hormonal responses. Journal of Applied Animal (2016). Effect of weaning age on growth Behaviour Science, 98: 70–88. doi: performance of cross breed pigs reared upto 70 10.1016/j.applanim.2005.08.014. kg body weight under intensive system. Journal Dema K (2016). Thai pigs to replace existing breed. of Animal Science and Veterinary Medicine, 1: Available at:
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pig rearing system. College of Natural Wangchuk R (2005). An integral part of Bongop Resources, Lobesa, Punakha. culture. Available at: Spreeuwenberg VMAM, Verdonk JMAJ, Bakker
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Full length paper Breed preference and breeding practices of dairy farmers in Bhutan
DEKI CHODEN* AND NAR B TAMANG
National Dairy Research Center, Department of Livestock, Yusipang, Thimphu.
*Author for correspondence: [email protected].
Copyright © 2018 Deki Choden. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: Cross breeding program started long ago but very little information has been documented. This study was conducted in all 20 districts with the objective to analyze breeding practices followed and breed preference by farmers. The study sampled 566 households spread across all four regions. Field interviews were conducted using a semi-structured open-ended questionnaire. Jersey with an exotic blood level of 62.5-81.25 percent was preferred mainly due to high milk yield and small body size requiring less feed. Both natural mating and Artificial Insemination (AI) were practiced. For breed improvement program, exotic breeding bulls were used since first Five Year Plan (1961-65), followed by AI in 1987. Farmers used AI more than other cross breeding methods. Along with improvement in genetic potential of animals, equal importance should also be given to factors such as good husbandry practices, appropriate genotypes suitable to different production systems, and proper recording system. This study suggests that there is a need for strategic investment and intervention by government to enable development of a dairy breed suitable to Bhutanese conditions. Further, a comparative study needs to be carried out to test whether Jersey outweighs other exotic dairy breeds in milk production and other parameters.
Keywords: Artificial Insemination; dairy; breed; cattle; cross breeding; milk.
1. INTRODUCTION 2002; Tamang and Perkins 2005). Jersey and Brown Swiss were two principal exotic breeds Dairy farming is widely practiced by Bhutanese introduced in the country for breed improvement farmers in the smallholder system. Farmers rear program when the first five-year development plan only dairy breeds for milk. The local breeds found started in 1961. Technical recommendations were in the country are Siri (Bos indicus), which is also to cross Thrabum with Jersey for mid to low called as “Thrabum” or “Nublang”. Siri breed is altitude areas while Brown Swiss crossbreeding used as base stock for developing composite breed was recommended for upper altitude areas. with exotic breeds. It is known to produce less milk However, upon farmers’ request, the breed barrier and has small body size. It requires less feed and is was lifted and the choice of breed was left open to highly adaptable to harsh environment, due to its farmers. Very recently, Holstein Friesian (HF) is sure footedness and ability to forage on steep also reared, especially in warmer southern belts terrain (Phangchung et al. 2002). Mithun and its after government allowed import of HF in 2014. crosses are also found in lower temperate and Ever since the first Five Year Plan (FYP) subtropical broadleaved forest (Phangchung et al. started, many programs and initiatives were put in 49
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 49-52, 2018 place to improve the local breed through Jersey, followed by Holstein Friesian (5.3%). crossbreeding with exotic breeds to increase milk Abundance of particular breed demonstrates the production. The breed improvement program in the farmers’ preference for that breed (Marius et at. early 1960s used exotic breeding bulls, which was 2011). While very few preferred Brown Swiss, followed by Artificial Insemination (AI) after the Thrabum and Mithun breeds, none of the institution of Semen Processing Centre (SPC) under respondents preferred buffaloes. Jersey is one of National Artificial Insemination Program (NAIP) in prominent dairy breeds found in countries where 1987. Crossbreeding these exotic breeds with cattle are primarily kept for milk production (Bebe Mithun crosses was not recommended. However, et at. 2003; Misganaw et al. 2014). although the breed improvement program started long time ago there is little information on breed 3.2 Preference of exotic blood level preferred and breeding practices followed by dairy About 33% of respondents preferred exotic blood farmer. Therefore, a study was conducted with the level slab 75-81.25%, followed by slab 62.5-75% main objective to understand the dairy breeds (29.5%) and 50-62.5% slab (14%) (Table 2). About preferred and breeding practices followed by dairy 11% of respondents opted for the higher slabs farmers. 81.25 and above. It shows that farmers have less preference for breeds with higher blood level, 2. MATERIAL AND METHOD although several crossbreeding experiments have demonstrated that crossbreds with high blood level 2.1 Study sites are undoubtedly better than native breeds (Singh Four regions namely Western, West-Central, East- 2016). It could also be because the yield in higher Central and Eastern, covering all 20 districts were crosses falls short of theoretical expectations. included in the study. The study sampled 566 Further, the total replacement of genes will not lead households spread across all four regions. Around to higher production in cattle. However, in the 90% of members from eight districts, two in each Indian context, exotic inheritance of around 50% is region were randomly interviewed, using a semi- most ideal for growth, reproduction and milk structured open-ended questionnaire. Where ever production. It is recommended that Holstein possible, informal discussions were held with some Friesian and Jersey inheritance with non-descript of the resourceful farmers to capture additional animals be maintained at around 50-62.5% exotic information on existing dairy breed improvement inheritance level for better production performance. program and breeding practices.
3. RESULTS AND DISCUSSION Table 2: Dairy cattle breed preference by blood level. 3.1 Breed composition and preference for dairy cattle breed Exotic blood level Frequency % Breed composition and preference for dairy cattle 50-62.5% 79 14.0 breed by farmers are presented in Table 1. The herd 62.5-75% 167 29.5 comprised of both crossbred and local animals. 75-81.25% 187 33.1 Amongst breeds, the highest proportion was Jersey 81.25-87.8% 67 11.9 (88.29%), followed by Mithun (6.12%) and 87.5 & above 65 11.5 Holstein Friesian (3.17%). Brown Swiss and Total 566 100 Thrabum were few in number. Although, Buffalo is found in the country, it was not reared by the respondents. In this study, from the total sample of 3.3 Reasons for breed preference 566 households, 521 respondents (92%) preferred Reasons for preference of cattle breeds are presented in Table 3. The preference for Jersey Table 1: Dairy cattle breed preference by dairy breed by over 71% of respondents was mainly due farmers. to high milk yield. The results are consistent with Breed Frequency % the findings of earlier studies in Africa where milk yield was ranked as top most preferred trait, Jersey 521 92 followed by growth rate, adaptation, breeding BS 5 0.9 ability, draught power, butter fat yield and coat Mithun 5 0.9 colours of the cow (Bebe et at. 2003; Misganaw et Thrabum 5 0.9 al. 2014; Marshall et al. 2016). Such a great HF 30 5.3 preference for high milk yield is common in cattle Buffalo 0 0.0 owners, who keep cattle primarily for milk Total 566 100 (Misganaw et al. 2014).
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Bhutanese dairy farming system is In this study, the most popular breeding characterized by scarce pasture, rough terrain and practice followed was AI (66.3%), followed by smallholder system. In such farming system, the natural mating with the use of government (42%) need for hardy breed that produces more milk and and private (22.6%) breeding bulls while CAIT had the dietary habits for liking butter might have not yet gained popularity. Beside breeding bull, AI determined farmers’ preference for a particular is also commonly used in India under the initiative breed. of National Dairy Development Board (NDDB). Over 28% of respondents’ preference for the AI, being a common breeding method in Bhutan, same breed was because the cattle are docile and could be attributed to widespread of AI facilities in easier to manage. Jersey is the smallest of dairy the country and increased accessibility to mobile AI breeds; they are easy keepers and performs better (Tshering pers. coms. 2017). Moreover, AI can be than Holsteins on rough scanty pastures; Jersey are done at farmers’ doorsteps, avoiding the need to general favorites as family cows on account of the make animal walk long distance for getting mated richness of their milk, high persistency of milking with breeding bull (NDDB, India). On the contrary, and their easy keeping quality and gentleness bull services (63%) outweighed AI (37%) in small (Anthony 2007). holders of Kenyan highlands (Bebe et al. 2003). The reasons for preferring Jersey suggests that when starting a herd, it is important to choose a 4. CONCLUSION breed of cattle most common in the community, High milk production, ability to perform better having average production of milk and fat (Eckles under scarce pasture, gentle and easy keeping and Anthony 2007). Bath et at. (1985) also qualities have led to preference for Jersey breed. mentioned that the most important consideration in The most preferred exotic blood line slabs are choosing a breed is the present and future milk between 62.5-75% and 75-81.25%. Natural mating market situation. by breeding bulls and AI, using frozen semen, are
the two breeding practices followed in the study 3.4 Breeding practices area. AI outweighs other breeding methods The breed improvement program in the country is followed by farmers. Respondents are of the view carried out by natural mating and crossbreeding that Jersey breed yields more milk than other dairy with the main objective to maximize exotic breed breeds. Nonetheless, further comparative study fraction in the herds. Natural mating is done by needs to be carried out to test whether Jersey government as well as private breeding bulls while outweighs other exotic dairy breeds in milk AI is carried out by the government AI technicians production and other parameters. Cross breeding is through Artificial Insemination Outreach Centres the most important component in dairy farming. (AIOC), using the imported or locally produced However, together with the improvement in genetic frozen semen. Breeding bulls are mostly Jersey and potential of the animals, it is equally important to few are Brown Swiss and Mithun, produced promote good husbandry practices and appropriate domestically in government farm. Private breeding genotypes suitable to different production systems. bulls are those genetically superior, which are fit for breeding. Very recently, to overcome the Acknowledgements shortage of AI technicians, early school leavers The authors would like to sincerely thank District were trained as Community Artificial Insemination Livestock Officers and officials from four Regional Technicians (CAIT) in some pockets who can Livestock Development Centers and Livestock provide AI services upon charging nominal service Extension Centers, for their unfailing support fee. Each subdistrict is provided with AIOCs and rendered to the team during the entire field work. breeding bulls at strategic locations. All the We also thank Mr. A.N. Koirala, Mr. Mohan Raj Table 3: Reasons for dairy cattle breed preference. Gurung and Mrs. Sangay Wangmo, for their assistance in field works. We also thank Mrs. Leela Reason Frequency % Maya Dahal and Mrs. Ganga Maya Rizal for their More milk production 526 71.4 assistance in data management. We are grateful to Easy to manage 210 28.5 all respondent farmers for sparing their valuable Fetch high price if sold 1 0.10 time and providing valuable information. Finally, we acknowledge the financial support, guidance and other logistic supports of NDRC, Yusipang. services provided by the government are free of cost except for the nominal service charges paid to REFERENCES CAIT.
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Bath DL, Dickenson FN, Tucker HA and Misganaw G, Wuletaw Z and Ayalew W (2014). Appleman RD (1985). Dairy Cattle: Principles, Breeding practices in indigenous dairy cattle Practices, problems, Profits. Washington: Lea breeds in Northern Amhara, Ethiopia. and Febiger. Livestock Research for Rural Development. Bebe BO, Udo H, Rowlands GJ and Thorpe W Available at: (2003). Small holder dairy systems in Kenya http://www.lrrd.org/lrrd26/4/misg26062.htm highlands: breed preference and breeding (Accessed on October 5, 2017) practices. Livestock production Science, 82: National Dairy Development Board (2016). Hand 117-127. Book of Good Dairy Husbandry Practices. Choden D, Timsina MP, Rai DB and Tamang NB Available at: (2017). Effects of government subsidy support https://www.slideshare.net/ashokkoori1/handbo on livilihood of dairy farmers in Bhutan. ok-ofgooddairyhusbandrypractices (Accessed Bhutan Journal of Animal Science, 1(1): 1-4. on September 22, 2017). Collado DM, Byrne TJ, Amer PR, Santos BS, National Dairy Research Centre (2013-2018). 11th Axford M and Pryce JE (2014). Analyzing the Five Year Plan documents. NDRC, Thimphu. heterogeneity of farmers preference for Phangchung, Dorji P, Sonam T and Peldon K improvements in dairy cow trait using farmer (2002). Sustainable development of dairy typologies. Journal of Dairy Science, 98: 4148- farming in Bhutan. ICIMOD, Katmandu, 4161. Nepal. DoL (2012-16). Livestock Statistics. Department of Sharma B and Banskota K (2002). Small Holder Livestock, Ministry of Agriculture & Forests, dairy farming in Nepal: Characteristics, Royal Government of Bhutan, Thimphu. Constraints and Development Opportunities. Eckles CH and Anthony EL (2007). Dairy Cattle ICIMOD, Katmandu. and Milk Production. Delhi: BIOTECH Singh C (2016). Cross-breeding in Cattle for Milk BOOKS. Production: Achievements, Challenges and International University of Business Agriculture Opportunities in India-A Review. Advances in and technology. (n.d.). Dairy production of Dairy Research, 4: 158. doi:10.4172/2329- different organizations of Bangladesh at a 888X.1000158. glance. Retrieved October 25th, 2017, from Tamang NB and Perkins JM (2005). Cattle LinkedIn Slieshare: https://www.slideshare.net/ management systems in humid subtropical Kahi AK (2002). Animal Genetic Training areas of western Bhutan. Journal of Bhutan Resource. Available at: http://agtr.ilri.cgiar.org studies, 2: 14-18. (Accessed on October 20, 2017). Wahome RG and Mwanyumba PM (2009). An Marius LN, Chikosbi EI, Mlambo BH and Mutisi C analysis of factors affecting smallholder mixed (2011). Breed preference, production farming activities, performance and performance and management of dairy cows intervention in Wundanyi location. Ministry of among selected small holder dairy farmers of Livestock Development, Department of Zimbawe. AGRICOLA. Veterinary Science. Nairobi: Private Bag- Marshall K, Wanyoike F, Ndiwa N, Ghebremaiam 000625. H, Mugumeri L et al. (2016). Traditional Wangchuk K, Wurzinger M, Darabant A, Gratzer G livestock breeding practices of men and women and Zollitsch W (2014). The Changing Face of Somali pastroralist: Preference and selection of Cattle Raising and Forest Grazing in the breeding animals. Animal breeding and Bhutan Himalaya. Mountain Research and Genetics, 4: 534-547. Development, 34 (2): 131-138.
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Full length paper Effects of poultry feed and stocking density on broiler performance
TASHI JAMTSHO* AND SURYA BC RAI
National Poultry Research and Development Centre, Department of Livestock, Ministry of Agriculture and Forests, Sarpang, Bhutan.
*Author for correspondence: [email protected].
Copyright © 2018 Tashi Jamtsho. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: This study was carried out with the objective to evaluate the effects of poultry feed under different stocking densities on Feed Conversion Ratio (FCR) and growth of broiler strain Ross 308. An experiment was conducted with three types of feed under three stocking densities. A total of 450 unsexed Day-Old Chicks (DOC) were used for the study. The types of feed tested were Karma feed, BMG feed and Samrat feed. The three stocking densities were low density (6 birds m-2), medium density (8 birds m-2) and high density (10 birds m-2). The areas allocated were 8.33 m-2, 6.25 m-2 and 5.0 m-2 for low, medium and high densities, respectively. Each area was stocked with 50 birds. Feed type showed a significant effect on the final live body weight. Mortality was not significantly affected by feed. At the end of experiment (42 days), the final live body weight was greatest for birds fed with Samrat feed in stocking density of 8 birds m-2. The lowest body weight was recorded for birds fed with BMG feed in stocking density of 10 birds m-2. The average final live weight across all stocking densities was recorded for Karma feed. The study results suggest that Samrat feed is the best among three feed brands for commercial broiler Ross-308.
Keywords: Broiler; feed brand; Feed Conversion Ratio; poultry; stocking density.
1. INTRODUCTION broiler production. In the recent years, with escalating feed cost in the market, profit from Poultry production is practiced worldwide and poultry has been declining every year (Vasanth et al. provides an acceptable form of animal protein, 2015). FCR determines the profitability and minerals and vitamins in human diet (Szőllősi et al. economic viability of broiler farm. FCR in broiler 2014). Broiler farming has been recognized as a production varies from region to region and farm to profitable enterprise and is much-preferred agro- farm due to different management practices. business than ruminants (Beg et al. 2011) with the According to Scott (2005), when feed intake is competitive edge that it can be harvested for human increased, the greater proportion of nutrients is used consumption within short time. According to for growth and better FCR is achieved. Samarakoon Shahimi et al. (2006), the important economical and Samarasinghe (2012) noted that FCR increases traits considered for broilers are growth rate, Feed with increasing age in both sexes of broiler from day Conversion Ratio (FCR), mortality and carcass 1 to 42. quality. Srivastava et al. (2013) reported that feed is In broiler production, Skrbic et al. (2009) stated a major component, affecting net return from that stocking density is a very important welfare poultry, as it accounts about 65 to 70% cost of 53
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 53-56, 2018 factor, which directly and indirectly influences and The areas allocated to low, medium and high determines the level of growth of chicken body stocking densities, were 8.33 m-2, 6.25 m-2 and 5.0 weight. Many studies on stocking density have m-2, respectively. Each area was stocked with 50 produced variable results in broiler production. birds. Some studies have shown great benefits under low On zero day, chicks were fed ad libitum and stocking density, while others revealed little or no from day 1 to 23, birds were fed with chick starter in differences. Tayeb et al. (2011 ) revealed no accordance with standard management guidelines of difference in live body weights and feed conversion Ross 308. On 24th day, 70% chick starter (crumble) ratio at 7 weeks of age under different stocking and 30% broiler finisher (mash) were fed. On the densities. following day, birds were provided 50% each of In Bhutan, poultry farming has gained starter and finisher. On 26th day, birds were fed with popularity and is one of the important livestock 30% starter and 70% finisher accordingly. From 27th components. Poultry development in Bhutan started day, the birds were provided with finisher till 42nd in the first five-year plan in 1961 with an aim to day. Birds were provided with 24 hours of lighting improve nutrition of rural population and alleviate on day one. In subsequent days, one-hour darkness poverty through increased egg and meat production. was provided till seven days of age. From 8th to 21st Further, it was intended to replace local birds by day, six hours darkness was provided and from 22nd exotic breeds to enhance production (Nidup et al. day, birds were provided with 12 hours of lighting 2005). Rearing of broiler chicken was not introduced till 42nd day. To avoid diseases entry into the in spite of poultry development because of the experimental site, all biosecurity measures were religious belief and other social stigmas (Nidup et al. incorporated and birds were vaccinated timely. 2005). The chicken meat demand was mainly met through import from neighboring states of India 2.3 Brooding of birds (Dahal 2007). With the increase in chicken The chicks were vaccinated against Marek disease at consumption, rearing of broilers has slowly gained the hatchery. Feed and water were provided ad momentum, especially in southern Bhutan. The libitum on day zero. From day 1 to 7, chick plates commonly used strain of broiler chicken is Ross- were used for feeding and round drinkers were used. 308. Although, Ross-308 is popular, FCR of Ross After 1 week, feeds were provided in round feeders 308 has never been studied. Therefore, a study was and heights of the feeders were adjusted according conducted with the objective evaluate the effects of to the height of birds. The average temperature and stocking density and feed type on growth and FCR humidity at birds’ level were 31.9°C Celsius and of commercial Ross-308. 78%, respectively. Fresh, clean and sundried saw dusts were used as bedding materials. Liquid 2. MATERIALS AND METHODS petroleum gas and electric brooders were used for brooding. 2.1 Experimental site
The experiment was conducted at National Poultry 2.4 Data collection Research and Development Centre, Sarpang district. The data was collected over a period of 42 days from The experimental area has wet tropical to humid 15th March to 26th April, 2017. The body weights subtropical type climate, characterized by hot were measured weekly and mortality was recorded summer and moderately cool winter. The average on daily basis. FCR, mortality and Average Daily annual temperature is 22oC and the average annual Gain (ADG) were determined on weekly basis by rainfall ranges from 1200 mm to 2500 mm. using the following formula.
2.2 Experimental design and treatments 푇표푡푎푙 푓푒푒푑 𝑖푛푡푎푘푒 (푔) A total of 450 unsexed Day-old Chicks (DOC) of 퐹퐶푅 = Ross-308 strain were used as experimental birds. 퐹𝑖푛푎푙 푤푒𝑖푔ℎ푡 푔푎𝑖푛 (푔) Three types of feed were applied across three 퐹𝑖푛푎푙 푤푒𝑖푔ℎ푡 − 퐼푛𝑖푡𝑖푎푙 푤푒𝑖푔ℎ푡 (푔) stocking densities. The feeds were Karma, BMG and ADG = Samrat feed. Each bird was an experimental unit 퐴푔푒 (퐷푎푦푠) from where measurements were recorded. Stocking density was not replicated. The conditions and 푁표. 표푓 푑푒푎푑 푏𝑖푟푑푠 푀표푟푡푎푙𝑖푡푦 = × 100 standards of the experiment were in accordance with 푁표. 표푓 𝑖푛𝑖푡𝑖푎푙 푏𝑖푟푑푠 the standard management guidelines of commercials Ross 308 (2015). The DOCs were randomly 2.5 Data analysis allocated to three-stocking density of 6 birds m-2 The data were entered in Microsoft Excel 2010. The (low stocking density), 8 birds m-2 (medium stocking normality of data was tested with Shapiro Wilk test density) and 10 birds m-2 (high stocking density). and homogeneity of variance was tested with Jamtsho and Rai (2018) 54
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 53-56, 2018
Levene’s test. Under each stocking density, the 1.72 at 42 days of age. The current study failed to effects of feed on FCR and growth of broiler were achieve this level of FCR. However, it is in tested with One-Way Analysis of Variance congruence with the result of Szőllősi et al. (2014) (ANOVA). The independent variable was feed and who observed final live weight of 2.59 kg with a the dependent variables were FCR and body weight. FCR of 1.88 at 42 days of age. Difference in dependent variables was considered significant when p values were below 0.05. The 3000 Karma BMG Samrat statistical software Statistical Package for Social Science (SPSS) version 23 was used for analyzing a a a a a the dataset. 2500 a
3. RESULTS AND DISCUSSION 2000 3.1 Growth rate At the start of experiment, there was no significant b b 1500 b difference in average body weight of broiler DoCs, distributed across different feed brands and stocking density. After 42 days, the birds fed with Karma and 1000 Samrat feeds had attained better marketable weight compared to birds fed with BMG feed (Figure 1). (g) weight body live Mean The marketable weights of birds were 2456.56 g and 500 2498.12 g for Karma and Samrat feed, respectively, compared to the body weight of 1448.04 g of birds 0 fed with BMG feed. Karma and Samrat feeds did not 6 birds/sq m 8 birds/sq m10 birds/sq m differ significantly in terms of body weight but their body weights were significantly higher than the Figure 1: Average live body weight of birds fed body weight produced by BMG feed. A study by with different feeds after 42 days. Means with Pathak et al. (2015) reported an average body weight different letters are significantly different at p≤0.01. of 1967.95±3.07 g up to 42 days, which is much lower than the body weights produced by Karma and 4.00 Samrat feeds. However, it is slightly higher than the Karma BMG Samrat body weight produced by BMG feed. The study 3.50 a revealed almost one kg live body weight difference a when fed with equal quantity of feed. Our findings suggest that Karma and Samrat feeds are of superior 3.00 a quality than BMG feed, although we did not analyze the quality of feeds. 2.50 b FCR b b b b b 3.2 Feed Conversion Ratio (FCR) 2.00 FCR was significantly affected by feed brand at 42 days of age (Figure 2). No significant difference in 1.50 FCR between Karma and Samrat feed was observed in all stocking densities. The final average FCR for 1.00 Samrat and Karma were 1.81 and 1.84, respectively. The lowest FCR of 2.82 was observed for BMG 0.50 feed. The results of this study on FCR are in agreement with the findings of Nidup and 0.00 Wangchuk (2007), who found better FCR with 6 birds/sq m 8 birds/sq m10 birds/sq m Samrat feed. A study by Zakeri et al. (2013) Figure 2: Effects of feed on FCR after 42 days revealed no significant difference in FCR among under three stocking densities. Means with different three dietary groups. Tayeb et al. (2011) also letters are significantly different at p≤0.01. revealed no significant difference in live body weights and FCR at 7 weeks of age under different 3.3 Mortality stocking densities. In contrast, Ullah et al. (2012) On an average, 4.67% (21) mortality was observed revealed significant difference in FCR among during the experiment. Of this, 4% mortality was different experimental diets. According to Aviagen recorded in the first week of brooding and 0.67% (2012), the standard in Ross 308 broiler, as-hatched occurred in the next 5 weeks. The mortality did not performance, is 2.77 kg live weight with FCR of Jamtsho and Rai (2018) 55
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 53-56, 2018 differ significantly between feed brands. This study Tayeb IT, Hassan SN, Mustafa MM, Sadeq SA, contradicts the findings of Ali et al. (2012) who Ameen GI and Hassan AM (2011). Effects of reported significant difference in mortality under various stocking density on productive different stocking densities and flock size. Further, performance and some physiological traits of a study by Hall (2001) showed significant increase broiler chicks. Research Opinions in Animal and in mortality with increase in stocking density. In veterinary Sciences, 1(2): 89-93. contrast, Ali et al. (2012) revealed increased Ullah MS, Pasha TN, Ali Z, Saima, Khattak FM and percentage of mortality with age of birds. Hayat Z (2012). Effects of different pre-starter diets on broiler performance, gastro intestinal 4. CONCLUSION tract morphometry and carcass yield. The Journal of Animal and Plant Sciences, 22 (3): The final body weight and FCR were comparable 570-575. between Samrat and Karma feed types. Feed types Vasanth S, Dipu MT, Mercy AD and Shyama K had no effect on mortality during the experiment. (2015). Studies on production performance. Samrat feed is the best among three feed brands for International Journal of Technical Research and commercial broiler Ross-308. Applications, 3 (3): 269-272.
Zakeri A, Chehraghi M and Taghinejad MR (2013). REFERENCE Effects of different feed forms on performance Ali MI, Azmal SA, Ali A and Faruque MO (2012). in broiler chickens. European Journal of Effect of density and flock size on growth Experimental Biology, 3(4): 66-70. performance of native chicken. Journal of Bangladesh Agricultural University, 10(1): 55– 59. Beg MA, Baqui MA, Sarker NR and Hossain MM (2011). Effect of Stocking Density and Feeding Regime on Performance of Broiler Chicken in Summer Season. International Journal of Poultry Science, 10 (5): 365-375. Hall LA (2001). The effect of stocking density on the welfare and behavior of broiler chickens reared commercially (Vol. 10). Animal Welfare. Nidup K and Wangchuk P (2007). Comparative study of broiler feeds in Bhutan. RNR Journal, 3(1): 13-14. Nidup K, Dorji P and Penjor (2005). A Reveiw of Poultry Development in Bhutan. (N. R. Institue, Ed.) Pathak R, Ali N, Kumar S and Chauhan HS (2015). Evaluation of Growth Performance of Broiler (COBB-400) under different composition of diets. International Journal of Life Science, 10 (4): 1465-1468. Samarakoon S M and Samarasinghe K (2012). Strategies to Improve the Cost Effectiveness of Broiler Production. Tropical Agricultural Research, 23 (4): 338– 346. Scott T A (2005). Variation in feed intake of broiler chickens. Recent Advances in Animal Nutrition in Australia, 15: 237-243. Skrbic Z, Pavlovski Z and Lukic M (2009). Stocking density – Factor of production, performance, quality and broiler welfare. Biotechnology in Animal Husbandry, 25 (5-6): 359-372. Srivastava BS, Niwas R, Singh PD and Bisen B (2013). Impact of herbal based diets on production efficiency of broiler. The Bioscan, 8(1): 119-122.
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Full length paper High fiber diet as molt inducers for commercial layers under deep litter system in Bhutan
TASHI JAMTSHO¹* AND NIDUP DORJI2
1District Livestock Extension, Department of livestock, Ministry of Agriculture and Forests, Paro, Bhutan 2Department of Animal Science, College of Natural Resources, Royal University of Bhutan, Lobesa, Punakha, Bhutan
*Author for correspondence: [email protected]
Copyright © 2018 Tashi Jamtsho. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: A study was conducted with the objective to evaluate the effects of high fiber diets as molt inducers on production performance of 72 weeks old Hyline Brown layers under deep litter system. The four treatment diets were layer ration, cracked yellow maize, whole oat grain and tapioca. A total of 108 hens were randomly assigned to four treatments. Each treatment was replicated three times and each treatment was allotted nine hens. High fiber diets (maize, oat, tapioca) were fed ad libitum with 8 hours of light during molting period of two weeks. After molting period, birds were then fed with layer diet ad libitum for next two weeks (recovery period), except for non-molted. The parameters measured were live body weight, egg production and mortality. A significant body weight loss was observed in tapioca molt group, followed by oat and maize. A complete egg cessation in tapioca and oat molt group was recorded on day 13. On day 5 and 7 post molt period, the birds in oat and tapioca molt group restarted laying egg, respectively. Hen day egg production was slightly higher for birds fed with maize than those hens fed with oat. On the other hand, egg production from tapioca fed birds was close to non-molted hens. The lowest hen mortality was in oat diet group, compared to other treatments. The study recommends the use of oat grain as molt inducer.
Keywords: Egg production; feed; high fiber diet; layer; molting; poultry.
1. INTRODUCTION solution to addressing the issue is molting. Molting is a periodic shedding and replacement of plumage, Poultry farming has gained momentum in Bhutan decrease in reproductive function and re-laying of over the recent years. Bhutan is self-sufficient in egg. Since 1930s, induced molting has been eggs (DoL 2014). However, poultry farmers are exploited in layer farms to maximize profits reluctant to cull spent hens because of social stigma (Gongruttananun et al. 2013; Bland et al. 2014). In and religious sentiment. Rearing old hens despite US, a poultry farmer who practice induced molting low production incurs loss to poultry farmers, which has been reported to make at least 30% higher profit may eventually discourage farmers from rearing than purchasing pullets (Bell 2003). poultry. Although, poultry farming is a success, there Traditionally, the total feed withdrawal or is an issue of spent layers being retained, causing restriction is a popular practice because it is simple, extra burden to poultry farmers. One promising less expensive and has a satisfactory post-molt 57
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 57-61, 2018 performance (Santos et al. 2014). However, this treatment was layer ration 27 as control, second method involves fasting and results in serious treatment was cracked yellow maize 27, third welfare issues as the birds are exposed to physical treatment was whole oat grain 27, and the fourth stress (Bland et al. 2014; Santos et al. 2014). In treatment was tapioca 27. Each treatment was addition, the complete feed withdrawal method replicated three times. Birds were housed in the same empties the gastrointestinal tract, which leads to shed but were partitioned with off-planks and Salmonella enteritidis infection in birds (Ricke et al. bamboos. The shed was divided into 12 2013). Alternatively, the use of high fiber diets was compartments. The size of each compartment was found successful in inducing molting in old layers 250 cm long, 100 cm high and 180 cm wide, to (Gongruttananun et al. 2013; Santos et al. 2014; accommodate nine birds per compartment. Feeders Bland et al. 2014) and prevent Salmonella enteritidis and drinkers were placed inside the compartments. colonization (Ricke et al. 2013). Although, such All three molt diets (maize, oats and tapioca) are alternatives are available, no research has been commonly grown by the Bhutanese farmers. The conducted in Bhutan to assess the effectiveness of diets were purchased from farmers. The fresh tapioca locally available fiber diets to initiate molt. tubers were peeled and chopped into fine cubic Therefore, the objective of this study was to evaluate particles, which were then sun-dried on clean and dry the fiber diets as viable molt inducers in poultry concrete floor for 3-4 days. Maize was cracked by farms in Bhutan. milling machine. The molt diets were analyzed for their basic nutrient content (Table 2). Neither 1. MATERIALS AND METHOD vitamins nor minerals were provided to the birds. Molting program was executed as per procedure 2.1 Rearing conditions Animal care protocols and experimental procedures of Gongruttananun et al. (2013) and Bland et al. in this feeding trial were approved by the Research (2014). We did not use feed additive. Non-molted Committee, College of Natural Resources, Royal birds were fed 120 g of layer ration and water ad University of Bhutan, on 4th September, 2015. Birds libitum and the lighting program of 16 hrs of light per were reared as per FAO standard of feed and space day was ensured throughout the experiment. High requirement for layers (1 m2 for 5 hens). Tungsten fiber diets and water were provided ad libitum for 14 bulbs (100-volt) were installed in the shed to meet days, which was the molting period. To regulate photoperiod of birds from day old chicks till culled targeted 8 hrs of photoperiod during molting period, (Hyline International 2014). 9 compartments were covered with cartons and Commercial complete rations based on age were opaque tarpaulins. We aimed to achieve complete provided to birds (Table 1). Clean and fresh water egg cessation within 14 days of induced molting was made available to birds at all times. Birds were period (Gongruttananun et al. 2013; Bland et al. vaccinated against Marek’s disease before the trial, 2014). After cessation of egg production, hens were Infectious Bursal disease on day 21 and 30, and fed with layer ration ad libitum for first two weeks, Newcastle disease on day 56 and 112. Moreover, and 120 g per bird per day were offered on disinfectant dip was placed in front of the shed. consecutive days. Lighting hours was increased by 30 mins in the first week, and then increased at the rate of one hour per week until 16 hrs photoperiod 2.2 Experimental design and molting program was attained. Seventy-two weeks old Hyline Brown birds were procured and reared under deep litter system at the 2.3 Data Collection poultry farm of College of Natural Resources. The To estimate the body weight loss, the hens were weighed at the beginning and at the end of the molting trial commenced for 140 days from February th to June 2016, including 14 days of induced molting. molting period on 14 day, using electronic A total of 108 spent layers were leg-tagged and weighing machine. Birds were weighed weekly for randomly assigned to four treatment diets. The first each treatment. Daily feed intake by bird was recorded to estimate egg feed ratio for each treatment. The egg feed ratio was calculated with the Table 1: Types of ration and nutrient content. following formula. Ration Crude Protein K cal kg-1 Ca (%) (CP) (%) 푃푟𝑖푐푒 표푏푡푎𝑖푛푒푑 푓푟표푚 푒푔푔푠 퐸푔푔 푓푒푒푑 푟푎푡𝑖표 = 퐶표푠푡 표푓 푓푒푒푑𝑖푛푔 ℎ푒푛푠 Starter 19.0 2,800 1 Grower 18.0 2,800 1 Layer 16.5 2,750 4
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Table 2: Nutrient content of different molt diets. Molt diet Dry matter Ash ODM Crude protein (%) Crude fat (%) Crude fiber (%) (%) (%) (%) Tapioca 87.80 1.86 98.14 3.86 9.1 15.63 Maize 87.34 1.08 98.92 6.81 9.97 7.95 Oat 89.17 3.21 96.79 12.56 14.3 22.35
Egg production and mortality were recorded daily 3.2 Egg cessation and relaying during molt and post-molt period. Prior to induced The day hens stopped laying egg and the day hens molting, egg production was recorded for two weeks started relaying egg after cessation for each to confirm pre-trial uniformity among different treatment diet are shown in Table 3. The lowest treatment diets. Egg production cessation and day of average body weight loss was observed in non- re-laying were also recorded. Hen day egg molted group at the end of the molting period. Birds production was estimated weekly with the following fed with oat and tapioca diet lost significant body formula. weight compared to maize and layer diet groups, which is similar to the finding of Gongruttananun et 퐻푒푛 푑푎푦 푒푔푔 푝푟표푑푢푐푡𝑖표푛 al. (2013). Birds fed with maize diet did not stop 푇표푡푎푙 푒푔푔 푙푎𝑖푑 𝑖푛 푎 푑푎푦 laying egg, which is similar to the finding of = x100 푇표푡푎푙 푛푢푚푏푒푟 표푓 ℎ푒푛푠 푝푟푒푠푒푛푡 푡ℎ푎푡 푑푎푦 Gongruttananun et al. (2013). Birds receiving tapioca diet stopped laying eggs on the 10th day of 2.4 Data Analysis feeding, while birds fed with oat stopped on 12th day. Dataset was tested for normality prior to analysis. On 13th day, all birds fed with tapioca and oat One-way ANOVA was used to test differences in stopped laying. measured parameters between treatment diets. At post-molt, all birds were fed ad libitum with Treatment diet was an independent variable. The layer diet for two weeks. The birds receiving tapioca dependent variables were body weight loss, post- diet consumed the highest layer feed per day, molt live body weight, feed intake, hen day egg followed by birds fed with oat. In the second week, production and egg weight. Differences between oat and tapioca group continued to consume treatment diets were considered significant when p comparatively higher layer feed than non-molted and values were less than 0.05. The entire dataset was maize group. The greater post-molt feed analyzed with SPSS version 23. consumption might be due to high body weight loss during the molting period. Tapioca feed assigned 2. RESULTS AND DISCUSSION birds lost higher body weight than those fed with oat. After molting, the hens in oat diet group restarted 3.1 Live body weight laying eggs on 5th day and hens in tapioca diet group The effect of different high fiber diets on the birds’ live body weights is presented in Figure 1. The 2500 Tapioca average body weight loss was higher for birds fed Oat with tapioca, followed by oat. The live body weights 2000 Maize of hens fed with tapioca and oat in our feeding trial Layer feed were close to 1573.2 g of body weight of birds fed with tapioca in Thailand (Gongruttananun et al. 1500 2013). From our results, tapioca and oat may effectively be used to induce molting because birds 1000 weighed within the recommended range of 1470g– 1570g after complete cessation of egg production Live body weight (gm) (Hyline International 2014). The higher body weight 500 loss of birds fed with tapioca and oat might be attributed to poor palatability as they have high fiber 0 content compared to maize. However, it is important Pre-molting Post-molting Weight loss to further estimate the hen day egg production and egg feed price ratio to confirm the claims made using Figure 1: Pre- and post-molting live body weights live body weight information. of hens fed with different diets.
Jamtsho and Dorji (2018) 59
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 57-61, 2018 on 7th day. Egg cessation in our study was slower explained by the adequate recovery feeding duration than the one reported by Aygun and Yetisir (2013) (Hassanien 2011) and thus, it suggests the and Gongruttananun et al. (2013). This could be due importance of adjusting feeding regime based on to slow body weight loss during molting period molt diets. (Hyline International 2014). 3.4 Mortality 3.3 Egg production The mortality among treatments was not The hen day egg production was used as an indicator significantly different (Figure 2). During molting of production performance of different molt diet 35 groups. Egg production from birds fed with high Layer feed fiber diets declined drastically in the second week of 30 Maize molting (Table 4). The average hen day egg Oat production is lower than the egg production of birds 25 reported by Gongruttananun et al. (2013). On the Tapioca other hand, the average hen day egg production in 20 tapioca diet group was higher than that reported by Gongruttananun et al. (2013). 15 Towards the end of post-molt period, diet Mortality (%) treatments did not differ significantly among each 10 other in mean hen day egg production. The highest 5 hen day egg production was in birds fed with maize, followed by oat diet group, tapioca diet group and 0 layer feed group. The egg production of birds fed Molt Post molt with tapioca and oat increased during post-molt period (Figure 2). Hens fed with oat ration laid Figure 2: Mortality during molting and post- slightly more eggs than tapioca group. This could be molting period.
Table 3: Body weight loss, feed intake per bird per day and egg cessation (mean±standard error). Means with different letters within columns differ significantly (p≤0.05). Treatment Body weight Day when egg Intake of layer ration after cessation of Day when egg loss (%) laying ceased egg laying laying restarted Week 1 Week 2 Layer feed 6.15±2.22 b Did not cease 120.00±6.20 a 120.00±9.19 a - Maize 8.44±1.09 b Did not cease 114.74±6.19 a 114.66±8.82 a - Oat 15.17±0.74 a 12 140.21±3.22 b 132.22±8.64 ab 5 Tapioca 18.32±0.99 a 10 145.74±1.20 b 136.53±1.15 b 7
Table 4: Hen day egg production for each treatment (mean±standard error). Treatment Non-molted Maize Oat Tapioca p value
Molting period Week 1 56.0±4.0 a 35.0±7.0 ab 18.0±8.0 b 40.0±10.0 ab ** Week 2 50.0±5.0 a 4.00±2.0 b 3.00±2.0 b 3.00±2.0 b *** Post-molt period Week 1 - 4 48.0±2.0 b 36.0±2.0 b 61.0±2.0 a 32.0±3.0 b *** Week 5 - 8 63.0±2.0 62.0±2.0 62.0±2.0 57.0±1.0 ns Week 9 - 12 62.0±2.0 b 78.0±2.0 a 74.0±1.0 a 63.0±2.0 b *** Week 13 - 16 60.0±2.0 c 79.0±2.0 a 67.0±1.0 b 71.0±1.0 b *** Week 17 -19 43.0±2.0 b 71.0±4.0 a 63.0±3.0 a 58.0±6.0 ab *** Average 56.0±2.0 65.0±4.0 62±4.0 57.0±4.0 ns **p≤0.01, ***p≤0.001, ns-nonsignificant
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Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 57-61, 2018 period, the mortality was higher for non-molted bird, Bland K, Utterback P, Koelkebeck K and Parsons C followed by those fed with tapioca and oats. There (2014). Evaluation of feeding various sources of was no mortality in maize-fed birds. Mortality of distillers dried grains with soluble in non-feed- birds in our study is similar to the findings of withdrawal moult programs for laying hens. Gongruttananun et al. (2013). Pecking amongst the Poult. Sci, 93 (6): 1421-1427. doi: birds appears to be the main cause of mortality in our 10.3382/ps.2013-03678. study. In the post molt period, the highest mortality Gongruttananun N, Guntapa P and Saengkudrua K was recorded for birds in tapioca diet group, (2013). The effects of a short-term moult method followed by maize and oat. The lowest mortality was using cassava meal, broken rice, or corn on found for birds in non-molt group. ovarian regression, bone integrity, and post- moult egg production and quality in older (95 3. CONCLUSION week) laying hens. Poultry Science, 92: 2798- 2807. doi: 10.3382/ps.2013-03151. The study findings show that oat can be effectively Hassanien HHM (2011). Effect of force moulting used as molting inducer. Although, hens fed with oat programs on egg production and quality of diet laid less number of egg, as compared to maize, laying hens. Asian Journal of Poultry Science, 5: the post-molt mortality was lowest. The study 13-20. doi: 10.3923/ajpsaj.2011.13.20. recommends inducing molting at younger age (less Hyline International (2016). Management guide. W- than 72 weeks of age) in order to achieve higher egg 36 commercial layers. Available at: production. http://www.hyline.com/UserDocs/Pages/36_CO
M_ENG.pdf (Accessed on 15 July 2016). Acknowledgements Livestock Statistics (2014). Livestock Statistics. The authors would like to thank the College of Department of Livestock, Ministry of Natural Resources for allowing them to conduct this Agriculture and Forests. Thimphu, Bhutan. research at the poultry farm. Ricke SC, Dunkley CS and Durant JA (2013). A
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Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 62-65, 2018
Full length paper effects of Total Mixed Ration (TMR) differ between progressive and less progressive dairy farms
WANGCHUK* AND JAMBAY GYELTSHEN
National Research and Development Center for Animal Nutrition, Department of Livestock, Ministry of Agriculture and Forest, Bumthang, Bhutan.
*Author for correspondence: [email protected]
Copyright © 2018 Wangchuk. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: A study was conducted with the objective to determine the effects of Total Mixed Ration (TMR) on milk yield and constituents. Feeding experiment was conducted at a progressive dairy farm in Phuntsholing and two less progressive farms in Sarpang. All cows in individual farms received same amount of TMR in addition to their normal diet. Milk yield was recorded before and after TMR feeding. Milk samples from all experiment animals were collected and analyzed weekly for nutrient composition. Feeding trial lasted for 21 days, including two weeks of adaptation period. There was no significant difference in milk production before and after feeding TMR at progressive dairy farm. However, milk production, fat, SNF and protein contents were significantly higher after TMR feeding at less progressive farms. The study findings suggest TMR as a healthy feeding strategy to improve nutritional status and enhance productivity of dairy cows. Where local feed ingredients are abundant, TMR feeding should be recommended for the dairy farms with poor feeding practices. The study also suggests TMR not to be recommended for commercial dairy farms with improved feeding practices. Formulation of TMR should be carried out under the supervision of trained extension personnel. Keywords: Dairy; milk; crude protein; Total Digestible Nutrients; Total Mixed Ration.
1. INTRODUCTION producers to improve productive efficiency of dairy cattle without incurring additional expenses. The Dairy farming is gaining momentum in Bhutan and high energy requirement of high yielding dairy cows the number of dairy farmers’ groups and has led to an increase in intake of concentrates. cooperatives has risen in recent years. The Royal However, the ration must possess appropriate Government has made concerted efforts to enhance quantity and quality of fibre with adequate particle milk production and various subsidies and market size in order to prevent nutritional and metabolic facilities are made available. Although, there are disorders, like displaced abomasum, laminitis, or improved cattle breeds, there is still a need to realize rumen acidosis (Zebeli et al. 2010). the full genetic potential of these breeds. One of the Shortage of feed resources in Bhutan usually important strategies to achieve this is to improve the occurs in winter when green pasture and fodder quality of feed, especially under farmers’ resources are not available. In some cases, the management condition. available feed resources are not fed in right amount, Feed constitutes about 70% of the total as per requirement of animal. This is one reason that production cost (Pond and Mishra 1981). The has not allowed dairy farmers to fully realize the competitive dairy industry is compelling milk 62
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 62-65, 2018 genetic potential of milking cows. Although, the farmers. The objective of the study was to evaluate compound cattle feed manufactured by commercial the effects of feeding TMR on milk yield and feed mills are available, majority of the backyard composition in dairy cows. dairy farmers at village level cannot afford to feed cattle concentrate to their cows. However, despite 2. MATERIALS AND METHODS feed shortage, milk production and productivity of 2.1 Study sites and selection of animals dairy cows can be improved substantially if the The study was conducted at Pasakha in Phuntsholing available feed resources are judiciously and and Gawathang in Sarpang where there is potential proportionately used. for dairy farming. In Phuntsholing, we selected a One of the relevant and useful feed formulation progressive dairy farm, whereas in Sarpang, we and fodder utilization technologies is the Total selected two less progressive dairy farms. Both study Mixed Ration (TMR) technology. TMR is a mixture sites experience sub-tropical climatic condition, of both roughages and processed ingredients, which is characterized by hot and humid summer formulated and mixed to supply animals with with moderately cold and dry winter. required amount of feed and nutrients, in a form that Six Jersey cross cows from progressive dairy precludes selection (Nissanka et al. 2010). The farm in Phuntsholing and five from backyard dairy benefits of TMR include increased milk production, farm in Sarpang were selected for the study. The enhanced use of low cost feed ingredients, ability to cows were in early lactation stage. All experimental control forage concentration ratio, lower incidence animals were subjected to only one TMR treatment. of metabolic and digestive disorder and reduced The health status of experiment animals was labour input for feeding (Chumpawadee and Pimpa inspected prior to commencement of feeding trial. 2009). According to Maekawa et al. (2002), feeding Body weights were recorded before and after the forage and concentrates separately results in cows experiment. consuming a high proportion of concentrates than intended, increasing the risk of ruminal acidosis. In 2.2 TMR preparation and application Bhutan, TMR is less popular and the technology is Feed ingredients and chemical composition of yet to be adopted by dairy farmers. Bhutan has experimental diets for respective farms are presented diverse fodder resources, which provide in Table 1 and 2. The green fodder and roughage opportunities to develop TMR technology within the were chopped to appropriate size before dairy farm. As a step towards developing TMR incorporating them into TMR diets. Based on body technology, we conducted a study at dairy farms weight, 16 kg TMR was fed to each cow at the managed by progressive and less progressive dairy progressive farm in Phuntsholing and 8 kg TMR to
Table 1: TMR formulation and chemical composition of the ingredients (B.B commercial dairy farm). Ingredients Composition (%) TDN(kg) DCP(kg) Ca (gm) P (gm) Fig 20 2.44 0.32 0 0 WBG 40 6.44 1.68 120 280 Maize grit 25 17.85 1.42 100 175 Molasses 3 1.2 0 0 0 Mustard oil cake 10 6.09 2.55 50 0 Mineral mixture 1 - - - - Salt 1 - - - - Source: Practical Guide for Feeding Dairy Cattle in Bhutan (2008)
Table 2: TMR formulation & chemical composition of the ingredients (Dairy backyard farm) Ingredients Composition (%) TDN(kg) DCP(kg) Ca (gm) P (gm)
Green fodder silage 55 8.58 0.99 55 55 DDG 10 11.29 0.85 15 105 Maize grit 20 10.71 1.42 60 105 Molasses 3 1.2 0 0 0 Mustard oil cake 10 6.09 2.55 40 56 Mineral mixture 1 - - - - Salt 1 - - - - Source: Practical Guide for Feeding Dairy Cattle in Bhutan (2008)
Wangchuk and Gyeltshen (2018) 63
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 62-65, 2018 each cow at the less progressive farms in Sarpang. 15 TMR was weighed daily and fed at 6.00 and 16 ns hours for 21 days. There were no refusals of TMR. 13 The roughage to concentrate ratios in TMR were
20:80 and 55:45 for progressive farm in 1 - 11 Phuntsholing and less progressive farms in Sarpang, respectively. 9
2.3 Milk yield measurement 7 Milk yield was recorded after two weeks of feeding st
TMR and continued till the 21 day. Milk samples cow (l) yield Milk 5 were collected weekly from all experimental animals and analyzed with the Milko-Tester machine for 3 milk protein, fat, SNF and lactose. 1 2.4 Data analysis Before feeding TMR After feeding TMR The data obtained from the field was processed in Figure 1: Milk production before and after feeding Microsoft Excel and analyzed, using the Statistical TMR in progressive dairy farm in Phuntsholing. Package for Social Science (SPSS) version 23. The paired sample t-tests were conducted to test the 10 statistical differences between before and after TMR Before feeding TMR feeding in respective farms. 9 After feeding TMR 8 3. RESULTS AND DISCUSSION 7 3.1 Milk yield and composition 6 In the dairy farm in Phuntsholing, there was no
% 5 significant difference in milk production before and after feeding TMR (Figure 1). Although statistically 4 non-significant, the SNF, lactose, and salt contents 3 were higher after feeding TMS (Figure 2). However, 2 fat content decreased after feeding TMR. The non- 1 significant difference in milk yield and constituents level is explained by the fact that the progressive 0 farm in Phuntsholing is a well-organized farm with SNF Fat Lactose Salt intensive management and improved feeding Figure 2: Milk constituents before and after practices. The farm is adjacent to the Pasakha feeding TMR in progressive dairy farm in Beverage factories and has access to Wet Brewery Phuntsholing. Grains (WBG), sold at cheaper rate. WBG is the feed that is fed separately. Whereas, the dairy farm major concentrate feeding source for the farm, along of Mr. Kinchab Loday is restricted to grazing and with high quality commercial concentrate feed. feeding waste slurry obtained from distillery. These WBG has low dry matter (20-32%), high protein separate feeding and poor quality of diet have led to content, and high content of total digestible nutrients low nutrient intake and thus, explains the consequent (TDN) as a result of high digestibility of the low milk production of dairy farms in Sarpang. available fiber (Chanie and Fievez 2017). Therefore, Moujahed et.al (2009) reported that the separate WBG are a good source of protein with a crude feeding of basal diet and concentrate results in protein that range from 25 to 34% (Thomas et al. concentrate wasting and frequent metabolic 2016). disorders such as acidosis, as a result of consuming Unlike the progressive farm in Phuntsholing, high amount of starchy concentrate. there were significant differences in both milk yield and constituents at the farms in Sarpang (Table 3). 4. CONCLUSION Milk yield, fat and SNF level increased significantly after feeding TMR. Similar result was reported by Where local feed ingredients are abundant, TMR Wachirapakorn et al. (1997) who observed increase feeding should be recommended for the dairy farms in milk yield after TMR feeding.The farms in with poor feeding practices. TMR is not Sarpang had been following different and poor recommended for commercial dairy farms with feeding system prior to TMR feeding. The normal improved feeding practices. Formulation of TMR animal diet of dairy farm of Mr. Pema Chojay is should be carried out under the supervision of mainly roughage, supplemented with concentrate trained extension personnel. Wangchuk and Gyeltshen (2018) 64
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 62-65, 2018
Table 3: Milk production and constituents before and after feeding TMR at less progressive farms in Sarpang. Farmer’s name Parameters Before 14 days after 21 days after Significance feeding TMR feeding TMR feeding TMR
Pem Chojay Milk (l) 4.61±0.21 b 6.70±1.18 a 6.83±1.11 a *** Fat (%) 3.77±0.09 b 4.90±0.31 a 5.37±0.41 a * SNF (%) 6.20±0.06 b 9.50±0.00 a 9.27±0.13 a ** Protein (%) 2.10±0.06 b 3.40±0.00 a 3.37±0.03 a ** Lactose (%) 3.37±0.08 b 5.07±0.09 a 5.07±0.08 a ** Salt (%) 0.50±0.00 a 0.70±0.00 a 0.70±0.00 a ns
Kinchab Loday Milk (l) 2.48±0.06 b 4.82±0.17 a 5.05±0.23 a *** Fat (%) 3.90±0.10 b 5.20±0.20 a 6.65±0.95 a ** SNF (%) 7.70±0.10 b 9.20±0.00 a 9.30±0.30 a ** Protein (%) 2.90±.10 b 3.30±0.00 a 3.30±0.00 a * Lactose (%) 4.50±0.10 a 5.00±0.00 a 4.90±0.00 a ns Salt (%) 0.60±0.00 a 0.70±0.00 a 0.70±0.00 a ns *p≤0.05 **p≤0.01 ***p≤0.001 ns: nonsignificant
Acknowledgement management on chewing activities, saliva The authors are very thankful to Dr. Kesang production and rumen PH of lactating cows. Wangchuk, RED, DoL, for guiding the entire Journal of Dairy Science, 85 (5): 1165-1175. research work, including statistical analysis. The Moujahed N, Dabbaoussi S, Amar BH and Darej S kind supports of Mr. Naiten Wangchuk (CLO), (2009). Effect of concentrate feeding strategy on RED, Dr. Kuenga Namgay (CFFO), AND, DoL, milk production and composition in dairy cows. Thimphu, Mr. Dorji Rinchen, Mr. Ugyen Dorji Research Journal of Dairy Science, 3 (1): 3-7. (DLO) Sarpang, Mr. Karma Wangchuk, Dr. Basant Nissanka NPC, Bandara RMA and Disnaka KGJ Sharma, RLDC Tsimasham and staff of NRCAN (2010). Comparative study feeding of Total Bumthang, are highly acknowledged. The authors Mixed Ration vs Conventional feeding on are grateful to Mr. Birkha Bdr. Vaisakha, Mr. Pema weight gain in weaned Friesian Heifers under Chojay and Mr. Kuenchap Loday, Gawathang, tropical environment. Journal of Agriculture Sarpang, for allowing their cows to be used for the Science, 5 (1): 56-67. experiment. Pond UK and Mishra M (1981). Feeding value and economics of mixed forages in milk production. REFERENCES Indian Journal of Dairy Science, 34: 49-53. Talib AA, Nassir A, Kashab AL and Almahdawi Chanie D and Fievez V (2017). Review on MK (2014). Effect of introducing dried brewers preservation and utilization of wet brewery Grains in diets of dairy cows on milk production spentgrain as concentrate replacement feed for and its composition. Euphrates Journal of lactating dairy cows. Journal of Animal Health Agriculture science, 6(3): 50-64. and production, 5 (1): 10. Thomas M, Hersom M, Thrift T, Yelich J (2016). Chumpawadee S and Pimpa O (2009). Effect of non- Wet Brewers’ Grains for Beef Cattle, Animal forage fiber sources in Total Mixed Ration on Sciences Department, UF/IFAS Extension, feed intake, nutrient digestibility, chewing University of Florida. behavior and ruminal fermentation in beef cattle. Wachirapakorn C, Puramongkol T, Seepuang V Journal of Animal and Veterinary Advance, 8 (1997). Total mixed Ration or Complete Mixed (10): 2038-2044. Ration for dairy cows. Journal of dairy cows, 5: Ibrahim MNM, Gyeltshen J, Tenzing K and Dorji T 53. (2008). Practical guide for feeding dairy cattle in Zebeli Q, Mansmann D, Ametaj BN, Steingasa H Bhutan. Department of Livestock, Ministry of and Drochner W (2010). A model to optimize Agriculture. the requirement of lactating dairy cows for Maekawa M, Beauchemin KA and Christensen DA physically effective neutral detergent fiber. (2002). Effect of concentrate level and feeding Archive of Animal Nutrition, 64: 265-268.
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Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 66-69, 2018
Full length paper Herbage yield and nutrient content of stampede oat under different irrigation intervals
DORJI WANGMO1* AND REKHA CHHETRI2
1Dzongkhag Livestock Office, Department of Livestock, Punakha. 2College of Natural Resources, Royal University of Bhutan, Lobesa, Punakha.
*Author for correspondence: [email protected]
Copyright © 2018 Dorji Wangmo. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: A study was conducted to assess herbage yield and nutrient content of oat cultivar Stampede under four different irrigation intervals. The trial was conducted at the Lobesa agricultural farm, College of Natural Resources (CNR), from 8th August, 2016 to 1st January 2017, over a period of 145 days, with 85 days to first cut and 60 days between first and second cut. Randomized complete block design was used as an experimental design with four replicates. The irrigation treatments were T1- rainfed as control, T2- irrigation after seven days, T3- irrigation after 14 days, and T4- irrigation after 21 days. Flooding method was used to irrigate the beds. In the first cut, the plants were significantly taller (87.45 cm) in T3 and the plants were shortest (69.41 cm) in T1. Maximum number of tillers (7.33) was observed in T2 and least number of tillers (3.43) was observed in T1. In the second cut, the plants were significantly taller (44.02 cm) in T3 and plants were shortest (35.25 cm) in T1. Tiller number was almost similar among irrigation treatments. It can be concluded that, under the existing environmental condition of Lobesa, T1 (irrigation at seven days interval) provides taller plants, more number of tillers per plant, high biomass yield and high crude protein content.
Keywords: Dry matter yield; forage oat; nutrient content; irrigation; stampede.
1. INTRODUCTION winter fodders, oats are said to be more palatable than wheat. Oats is a multi-cut crop and green oats Shortage of green forages during dry winter months is reported to yield double than the green wheat and early spring is a major constraint affecting the (Gyaltshen et al. 2009). Oats can be intercropped productivity of livestock. Fodder scarcity problem with other agricultural crops. Among oat varieties is severe from January to April. The only fodder tested in Bhutan, oat cultivar Stampede was found resources available during this period are wheat suitable for enhancing fodder production during straw, paddy straw and grain products, which are lean season. almost devoid of nutritional values and biomass Water is an essential factor for every (Gyaltshen 2002). developmental phase, starting from seed Oat (Avena sativa) is an important forage crop germination to maturation of a crop. Availability of introduced in Bhutan (Gyaltshen 2002). Many oat adequate amount of moisture at critical stages of varieties were also introduced for grain production, plant determines the productivity of any crop but farmers adopted oats as fodder (RNRR-RC (Jamal et al. 1996). Water stress during crop growth Jakar 2008). Traditionally, oat is grown after paddy leads to reduction in total biomass. Therefore, the harvest and is used as winter fodder. Among green accurate evaluation of crop water requirement is 66
Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 66-69, 2018 essential for increased oat production. Studies have 2.3 Management and treatment application investigated the effects of irrigation interval on About 1.50 kg cow dung was applied to each plot. yield performances of cereal crops (Khan 2000; Seeds were sown in lines at the rate of 30 kg ac-1. Sasani et al. 2004) and irrigation interval was found Spacings followed were: 20 cm between lines, 100 to have a significant effect on yield components cm between replications and 50 cm between plots. and qualitative traits of sorghum (Moosavi et al. The amount of water applied to each plot was 443.5 2011). However, irrigation is expensive and it is ml while the control plot (rain fed) did not receive important to identify accurate irrigation interval any irrigation. Weeding of trial plots was done as that produces optimum forage yield and quality of and when required. oat. Although, oat is extensively used as winter forage in Bhutan, little is known on the effects of 2.4 Field measurements irrigation interval on oat yield and quality. Plant height, number of tillers, plant density and Therefore, a study was conducted to evaluate the biomass (BM) yield were measured at booting potential of oat variety Stampede to provide stage during first cutting (85 days). Similar optimum forage yield and quality under different measurements were carried out during the second irrigation intervals. cut i.e. 60 days after the first cut. Twenty plants were randomly selected and their heights and tiller 2. MATERIALS AND METHODS number were measured. The plants were then harvested and fresh biomass was measured. A 2.1 Study area frame (0.50m×0.50m) was randomly placed four The agriculture farm of College of Natural times per plot to obtain yield from 1 m2. Plant Resources, Lobesa, Bhutan, was selected as a study materials inside the frames were harvested and site. It is situated at an altitude of 1,450 meters bulked. The plant materials were then thoroughly above sea level (masl). The area falls under dry mixed and a representative sample weighing 100 sub-tropical climate, which experiences hot gm was collected for dry matter and nutrient summer and cool winter. The mean maximum analysis. Samples were analyzed for dry matter annual rainfall of 4490 mm is recorded in content, crude protein, crude fiber, moisture, ash September and the mean minimum of 30 mm in and ether extract. November. The site experiences mean maximum temperature of 31.66oC, minimum temperature of 2.5 Data analysis 6.31oC and relative humidity of 81.50%. The soil is The data was entered in Microsoft Excel reddish clay, which is slightly acidic (pH=6.32) spreadsheet and exported to SPSS. Multivariate with low nitrogen content (0.03%), organic carbon ANOVA was used to analyze the data. Difference (0.4%) and moisture content (4.17%). between treatment means was considered
significant when p value was less than 0.05. The 2.2 Experimental design and treatment entire dataset was analyzed with statistical software The field experiment was carried out from 18th SPSS version 23. August 2016 to 1st January, 2017. Randomized complete block (RCB) was the experimental design 3. RESULTS AND DISCUSSION and irrigation interval was the treatment. The four treatments were; T1- rainfed as control, T2- 3.1 Plant growth irrigation after seven days, T3- irrigation after 14 Plant height and tiller number days, and T4- irrigation after 21 days. Each The mean plant height for first and second cut is treatment was replicated four times. Individual plot presented in Table 1. The overall average plant size was 3.6 m2 (3m×1.2m). height was 81.08 cm in the first cut and 40.70 cm in Table 1: Mean plant height and tiller number in first and second cut under different irrigation treatment. First Cut Second Cut Sample Mean plant Number of Mean plant Number of Irrigation treatment size height (cm) tillers height (cm) tillers
a) Rain fed 80 69.41 3.43 35.25 2.30 b) Irrigation after 7 days 80 87.31 7.33 36.90 2.23 c) Irrigation after 14 days 80 87.45 5.43 44.02 2.33 d) Irrigation after 21 days 80 80.14 4.48 37.39 2.33 Total 320 324.31 5.16 38.39 2.29
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Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 66-69, 2018 the second cut. In the first cut, the tallest plant observed in T3 and lowest in T1 (1418.3 plants m- height (87.45 cm) was obtained from T3 plot 2). In the second cut, the highest plant density (irrigation after 14 days), followed by T2 plot (3694.50 plants m-2) was observed in T4 and lowest (87.31 cm) (irrigation after 7 days) and T4 plot (2421 plants m-2) in T1. (80.14 cm) (irrigation after 21 days). The shortest In the first cut, the overall average biomass plant height was obtained from T1 plot (69.41 cm) yield obtained was 10.30 kg. In the second cut, the (rainfed). Similarly, in the second cut, the tallest average biomass yield was 1.49 kg. Biomass yield plant height (44.02 cm) was obtained from T3 plot was highest (11.97 kg) in T2 and lowest (6.54 kg) and the shortest (35.24 cm) from T1 plot. The result in T1. Among treatments, T2 gave the maximum agrees with the findings of Ahmad et al. (2014), biomass yield in the first cut. In the second cut, the who reported maximum plant height of 80.91 cm highest biomass yield (1.97 kg) was obtained from under irrigation at 20 days interval and the shortest T4. plant height of 64.46 cm under irrigations at 40 and The overall mean dry matter yield produced 60 days intervals. was 0.92 kg in the first cut and 1.74 kg in the The second cut result is in line with the second cut. In both the cuts, the highest dry matter findings of Gyaltshen (2002), who obtained yield was 0.27 kg and 2.19 kg produced from rain maximum plant height of 46.35 cm (average of fed irrigation. The lowest dry matter yield was three cuts). The overall results are in agreement obtained from irrigation after 7 days (0.15 kg). with the findings of Tahir et al. (2014) that plant A significant difference among treatments in height is significantly influenced by irrigation at dry matter yeild was observed in both first and different critical crop growth stages. The results second cuts. However, the dry matter yeild suggest that long irrigation interval beyond three increased during the second cut by 1.55 kg. The weeks is not favorable for plant growth. increase in dry matter yield was observed at The overall average number of tillers per clump different maturing stages. Kirilov (2004) and was 5.16 in the first cut and 2.29 in the second cut. Mohammad et al. (2004) reported that dry matter The maximum number of tillers per clump (7.33) yield increases with the advancing maturity. was recorded in T2 and minimum number of tillers per clump (3.43) in T1. This result is similar to the 3.3 Forage quality findings of Mohammad et al. (2004) who obtained Crude protein, ash and crude fiber highest number of tillers per plant (7.17) after 85 Table 3 presents the nutrient content of oat, mainly days of sowing. The variation in the number of crude protein, ash and crude fiber, under different tillers per plant may be due to difference in irrigation treatments. The overall average crude irrigations intervals. The study revealed that water protein content was 9.38% in the first cut and is very important for the development of tillers as 13.34% in the second cut. In the first cut, the the treatment with shorter irrigation interval had the highest crude protein content (9.99%) was observed maximum number of tillers in the first cut. in T3 and the lowest crude protein content (8.39%) Significant difference was observed in the number in T2. On the contrary, in the second cut, the of tillers per clump during first cut, however, only highest crude protein content (14.17%) was small difference was observed in the second cut. observed in T2 and the lowest crude protein content (12.19%) in T3. The result is in agreement with the 3.2 Forage yield findings of Tahir et al. (2014) that different levels Plant density, biomass and dry matter of irrigation significantly affect crude protein Table 2 presents the plant density, biomass and dry content. Tahir et al. (2014) also reported maximum matter of oat under different irrigation treatments. crude protein (9.13%) in plants that received single The overall average plant density was 1445 in the irrigation 21 days after sowing and minimum crude first cut and 2846 in the second cut. In the first cut, protein of 8.41% was observed in plants with no the highest plant density (1701.5 plants m-2) was irrigation. Table 2: Mean biomass yield from two cuts. First cut Second cut Irrigation method Mean Biomass SD Mean Biomass SD a) Rain fed 6.54 0.63 1.15 0.18 b) Irrigation after 7 days 11.97 0.38 1.26 0.13 c) Irrigation after 14 days 11.85 0.34 1.61 0.36 d) Irrigation after 21 days 10.84 0.84 1.96 0.38 Total 10.30 2.33 1.49 0.42
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Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 66-69, 2018 The average ash content was 9.83% in the first South Asia Pro Poor Livestock Policy cut and 18.23% in the second cut. The highest ash Programme. BHGP11: 1-6. content of 11.03% in the first cut was observed in Hussain S, Rehman M, Khaliq T, Haq A and Tahir T4 and the highest ash content of 21.25% in the MG (2014). Effect of different irrigation levels second cut was in T3. The lowest ash content of on yield and forage quality of oat (Avena 9.06% in the first cut was observed in T2 and the Sativa), Applied Science Report, 3(1): 2-6 lowest ash content of 15.83% in the second cut was Jamal M, Nazir MS, Shah SH, Nazir A (1996). observed in T1. Similar results were obtained by Varietal response of wheat to stress at different Hussain et al. (2014) who reported the maximum growth stages and effect on grain yield, straw total ash under frequent irrigation. Lack of yield, harvest index and protein contents in irrigation during critical growth stage negatively grains. Rachis, 15: 38-45. influences the oat growth, which leads to decline in Khan NA (2000). Simulation of wheat growth and forage yield and quality. yield under variable sowing dates and seeding In the first cut, the highest crude fiber content rates. M.Sc. Thesis, Dept. of Agron., Univ. of 1.62% was observed in T4 and lowest of 0.92% Agric., Faisalabad. in T3. In the second cut, the highest crude fiber Kirilov A (2004). Fodder Oats in Europe. Fodder content of 19.13% was observed in T2 and lowest oats: a world overview. Food and Agriculture fiber content of 18.79% in T3. The result showed Organization of the United Nation, Rome. that crude fiber increased during the second cut. Mohammad D, Khan S and Hussain A (2004). The crude fiber content in plant increases with Clipping of oats at various intervals on herbage maturity. The results agree with the findings of yield, forage quality and seed rate, Pakistan Mohammad et al. (2004) who reported similar Journal of Agricultural Marketing. 18(1): 72-75 results for oat. Moosavi GRS, Seghatoleslami MJ, Javadi H and
Table 3: Crude protein, ash and crude fiber content of oat for first and second cut under different irrigation treatment. First cut Second cut Irrigation treatment CP% Ash% CF% CP% Ash% CF%
a) Rain fed 9.63 9.41 1.33 13.86 15.83 19.08 b) Irrigation after 7 days 8.39 9.06 1.24 14.17 19.25 19.13 c) Irrigation after 14 days 9.99 9.81 0.92 12.19 21.25 18.79 d) Irrigation after 21 days 9.52 11.03 1.62 13.13 16.62 18.87 Total 9.38 9.83 1.28 13.34 18.24 18.97
4. CONCLUSION Ansari-nia E (2011). Effect of irrigation intervals and planting patterns on yield and This study demonstrated that short irrigation qualitative traits of forage sorghum. Adv interval provides better plant growth, high forage Environ Biol 5:3363-3368. yield and better forage quality. No irrigation or RNR-RC Jakar. (2008). Forage Production, a long irrigation interval gives higher yield but of low handbook for feed and fodder development forage quality. worker in Bhutan. 2nd ed. Renewable Natural Resources Research Center, Bumthang. REFERENCES Bhutan. Gyaltshen T (2002). Experiences with oats (Avena Sasani S, Jahansooz MR and Ahmadi A (2004). sativa) at temperate and high elevations of The effects of deficit irrigation on Water-use Bhutan. Proceedings of the fifth meeting of the efficiency, yield, and quality of forage pearl Temperate Asia Pasture and Fodder Network millet Proceedings of the 4th International Crop (TAPAFON). Renewable Natural Resources Sci Congress Brisbane, Australia, 26 Sep-1 Research Centre, Bajo, Bhutan. Oct. Gyaltsen T, Nirola H, Dorji Y, Thinley G and Tahir GM, Haq AU, Khaliq T, Rehman M and Tshering N (2009). Promoting a Productive & Hussain S (2014). Effect of Different Irrigation Sustainable Fodder Programme: Oats as a Levels on Yield and Forage Quality of Oat Potential High-Altitude Winter Fodder Crop. (Avena Sativa). Applied Science Report, 7(1): 42-46.
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Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 70-74, 2018
Full length paper Dry matter content of fodder resources utilized by dairy farmers’ groups in West-Central Bhutan
AITA K BHUJEL*, UGYEN NAMGYEL AND BIR D RAI
Regional Livestock Development Center, Department of Livestock, Ministry of Agriculture and Forests, Wangdue, Bhutan.
*Author for correspondence: [email protected]
Copyright © 2018 Aita K Bhujel. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: The study objective was to evaluate nutrient and Dry Matter (DM) content of available fodder resources utilized by Dairy Farmers’ Groups (DFG) in the west-central region. Two DFGs were randomly selected from each district in the region. Samples of available fodders were collected from DFGs. The available fodder resources, including local grasses, improved pasture and the fodder trees were listed. The samples were collected in early spring during the month of March and April in 2017 when fodder plants were flushing and tender. Hundred and ten fodder samples were collected and dried in the laboratory for DM analysis. Secondary data were collected from farmers. The results indicated that farmers used maximum of 41.61% fodder trees as source of fodder to feed their dairy cattle. The mean DM content of available fodder differed significantly. The maximum DM content of 27.68% was in tree fodders and minimum DM content of 19.60% in improved pasture. Overall, the Dry Matter Intake (DMI) of dairy cattle was 8.46 kg, which indicated that the requirement is within the range required by a dairy cow in the region, taking into consideration the average live weight of cattle as 300 kg. The findings of this study on the available fodder resources and the DMI of dairy cattle in the west-central region revealed that there are adequate fodder resources available to meet the DM requirement of dairy cattle.
Keywords: Dry matter; fodder resources; fodder tree; local fodder; improved pasture.
1. INTRODUCTION may increase up to 5% of body weight, and even higher in extremely high-producing cows. The The year-round availability of fodder is important to typical peak DMI values are in the range of 3.5%– sustain dairy development. Dry matter (DM) is an 4% of body weight (Herdt 2016). The reliable important component, which determines the estimates of forage DM content can help managers chemical composition and nutritive value of feeds to achieve the objectives of forage conservation, and fodder. Livestock, particularly the dairy cattle, livestock feeding and pasture allocation. needs to consume certain amount of DM in a day. DM content in fodder plants increases with On an average, about 10-11 kg DM day-1 is required maturity. DM is an indicator of the amount of by a cow to maintain health and production. Dry nutrients that are available in particular feed. The Matter Intake (DMI) is affected by both animal and knowledge on DM in feed helps to understand the feed factors. Body size, milk production, and stage amount of DM an animal can obtain from the of lactation or gestation are the major animal factors. feedstuff. It also helps to determine the amount of During peak, the daily DMI of high-producing cows concentrate feed that is required as supplement. 70
Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 70-74, 2018 However, in Bhutan, the nutritional of mid canopy and tree top. Upon collection of fresh requirements of dairy cattle for optimum production samples, the leaves were cleaned to remove any are less emphasized, particularly the nutritional visible surface contaminants and then weighed. The content and quality improvement of available fodder fresh samples weighing 200 to 500g were collected resources. Therefore, a brief study was conducted from each fodder species. A total of 110 fresh fodder with the Dairy Farmers Groups (DFGs) in the west- samples (68.32%) were collected from five districts. central region on available local fodder resources The weight of fresh samples was recorded in the and the amount of DM fed to the dairy cattle. The field, using a digital weighing balance. The samples objectives were to assess the DM content of were dried in hot air oven to determine DM content. available fodder resources and recommend feeding The number of samples collected from each district regime for effective dairy production. is presented in Figure 2. The highest number of
2. MATERIALS AND METHOD 2.1 Study sites and sample collection The districts and study sites of west-central region are presented in Figure 1. The west-central region has 30 functional DFGs. Among them, two DFGs were randomly selected from each district. Samples of available fodder were collected from these two DFGs. The DFG members were asked to list down the available fodder resources that included local grasses, improved pasture and fodder trees. Most of the available fodder species that reappeared within and among DFGs were excluded. The average daily intake of fresh fodder by cattle was collected by interviewing farmers through recall method. The amount of local fodder consumed by the cattle was estimated based on the amount of time spent while grazing during the day. The samples were collected in early spring during March and April in 2017 when fodder plants were flushing and tender. The plants were identified using books and published articles. A minimum of 10 random samples of fodder preferred by farmers were collected from each DFG. The grass samples were cleaned and cut into appropriate sizes and its fresh weight was measured. The procedure of Upreti and Shrestha (2006) for fodder tree sampling was followed. Samples were collected from all four sides Figure 1: Study sites.
12 11 Local fodder Improved fodder Fodder Trees 10 10 8 8 8 8 8 7 7 7 7 6 6 6 6 6 5
4
No. of Samples collected Samples of No. 2
0 Dagana Tsirang Gasa Punakha Wangdue -District- Figure 2: Total number of samples collected from study districts.
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Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 70-74, 2018 samples was collected from Dagana and the lowest dry sample weight. The highest dry sample weight was collected from Gasa (Figure 2). The samples in fodder tree could be attributed to leaf stalks and were oven dried in paper bags at about 105oC for 24 branches in the sample. hours. The weights of dried samples were measured The DM content varied significantly (p≤0.001) to determine the DM content of fodder species. The among different types of fodder. In general, the results were entered in Microsoft Excel and analyzed feeding of mixture of all three types of available with SPSS version 16. fodder could provide 22.82% DM to the dairy cattle in the region. Among the types of fodder available, 3. RESULTS AND DISCUSSION fodder tree contributes maximum DM of 27.68% and improved pasture contributes minimum DM of 3.1 Available fodder resources 19.60% (Table 3). Local fodder and improved Generally, there were around 161 fodder species pasture were moderately low in DM content as available and used by DFG members for feeding compared to tree fodder. This could be attributed to dairy cattle in the region (Table 1). The fodder the stage of fodder growth since the fodder samples resources available were mainly local fodder, were collected during the vegetative stage. improved pasture and tree fodder. Tsirang district Therefore, it is important to consider this fact while had a maximum number of 47 types of fodder formulating feeding regimes and diet for dairy cattle. resources. The least was in Gasa district with 17 types of resources. Among the available fodder 3.4 DM Requirement and Intake resources, fodder tree was utilized more (41.61%), The mean DMIs of DFGs are presented in Table 3. followed by local fodder (37.89%) and improved The mean DMI was 8.46 kg per animal. The highest pasture (20.50%) (Table 1). DMI was in Goserling DFG, Tsirang and the average Table 1: Number of available fodder resources in DMI was 9.35 kg. The lowest was in Damji/Khatoe, five districts in west-central region. Gasa with mean DMI of 7.78 kg. The average DMI required by each DFG in the region was 7.5 – 9 kg, Number of fodder resources considering the standard live body weight of 300 kg District Local Improved Fodder Total for Bhutanese cattle (Samdrup et al. 2010; fodder fodder Trees Wangchuk et al. 2017). The DMI requirement of adult cattle feeding on Dagana 10 6 20 46 average quality pasture is 2.5–3% of body weight Tsirang 18 8 21 47 (http://pir.sa.gov.au). The DMI of this study is Gasa 6 4 7 17 within the range and it is meeting the requirement of Punakha 10 9 9 28 dairy cows. However, the DM content varies across Wangdue 17 6 10 33 different seasons.
4. CONCLUSION 3.2 Common fodders available in the region In fodder tree category, the common fodder tree There is a need for similar studies for different species were Ficus roxburghii, Ficus cunia, seasons and stages of fodder growth to derive Bauhinia purpurea, Ficus lacor, Grewia tiliaefolia, complete conclusion of DM content of available Ficus nemoralis and Ficus benjamina L. In fodder resources in the region. There is also a need improved fodder category, the common species were for analysis of other nutrient contents such as crude Brachiaria ruziziensis, Setaria sphacelata, protein, neutral detergent fiber and energy. Adequate Pennisetum purpureum, Tripsacum laxum, laboratory facilities are needed at regional level Pennisetum clandestinum, Trifolium repens and beside the facility at the National Research and Avena sativa. Similarly, in local fodder category, the Development Center for Animal Nutrition. In order common species were Thysanolaena maxima, to balance DMI of dairy cattle round the year, the Cynodon dactylon, Setaria pallidesesca, Persicaria DFG members are recommended to practice fodder nepalensis, Bidens pilosa and Galinsoga parviflora. conservation (hay and silage) during fodder abundance season and use them during the fodder 3.3 Dry Matter analysis deficit period. Considering that the fodder trees The dried sample weights of three different types of provide maximum DM, it is important to encourage available fodders are presented in Table 2. There farmers to plant more fodder trees. was a significant difference (p≤0.05) among fodder categories in dry sample weight. The highest dry REFERENCES sample weight was found in fodder tree (90.99g), Calculating Dry Matter Intakes for Various Classes followed by improved pasture (69.58g) and lowest of Stock. Available at: www.pir.sa.gov.au, in local fodder (68.18g). Fodder tree had the highest (Accessed on 17 July, 2017). Bhujel et al. (2018) 72
Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 70-74, 2018 Dangol DR (2005). Species composition, Parker C (1992). Weeds of Bhutan. National Plant distribution, life forms and folk nomenclature of Protection Center, Simtokha, Department of forest and common land plants of western Agriculture. Sayce Publishing, 57 Marlborough, Chitwan, Nepal. Institute of Agriculture and St Leonards, Exeter, United Kingdom. Animal Sciences, Rampur, Chitwan, Nepal Samdrup T, Udo HJM, Eilers CHAM, Ibrahim Journal of Agriculture and Animal Science, 26: MNM, van der Zijpp AJ (2010). Crossbreeding 93-105. and intensification of smallholder crop-cattle farming systems in Bhutan. Livestock DoL (2016). Livestock Statistics 2016. 11th Series, Science,132:126-134. Department of Livestock, Ministry of doi:10.1016/j.domaniend.2008.06.005. Agriculture and Forests. Thinley U (2004). Know the plants of Bhutan. Ghosh C, Das AP (2011). Some useful and Volume – I, 2nd Edition. Thimphu, Bhutan. poisonous tea garden weeds from the Darjeeling Thomas TH (2016). Nutritional Requirements of District of West Bengal, India. East Himalayan Dairy Cattle. Department of Large Animal Society for Spermatophyte Taxonomy. Pleione, Clinical Sciences and Diagnostic Center for 5(1): 91-114. ISSN: 0973-9467.
Table 2: Average DM content of different types of available fodder across DFGs in west-central region. District DFG Fodder Type No. of samples Mean DM% SD CV% Dagana Zingchela, Local Fodder 4 15.76 4.10 26.04 Tsangkha Improved Pasture 3 17.74 1.87 10.57 Fodder Tree 6 29.57 8.20 27.73 Tashiding Local Fodder 2 15.22 3.94 25.88 Improved Pasture 4 19.60 5.35 27.32 Fodder Tree 5 25.22 5.61 22.25
Tsirang Goserling Local Fodder 6 27.52 12.23 44.45 Improved Pasture 2 22.28 0.59 2.65 Fodder Tree 3 27.42 5.69 20.75 Patshaling Local Fodder 2 12.04 3.91 32.47 Improved Pasture 4 17.02 3.34 19.62 Fodder Tree 5 25.36 3.84 15.16
Gasa Damji/Khatoe Local Fodder 2 9.69 3.09 31.88 Improved Pasture 4 17.20 4.98 28.93 Fodder Tree 3 29.03 10.18 35.08 Yamina/Khailo Local Fodder 4 13.04 3.97 30.45 Improved Pasture 2 17.17 10.09 58.76 Fodder Tree 4 24.89 6.41 25.76
Punakha Kabjisa Local Fodder 4 23.32 8.99 38.55 Improved Pasture 5 20.28 6.02 29.69 Fodder Tree 2 22.45 3.74 16.67 Toepaisa Local Fodder 3 12.72 5.78 45.46 Improved Pasture 3 23.96 3.58 14.94 Fodder Tree 5 20.09 5.99 29.82
Wangdue Busa, Sephu Local Fodder 5 22.86 13.51 59.12 Improved Pasture 3 18.44 4.99 27.06 Fodder Tree 3 46.51 15.65 33.64 Bajo, Tsedtsho Local Fodder 5 29.67 26.48 89.24 Improved Pasture 2 25.69 7.06 27.46 Fodder Tree 5 30.16 6.87 22.79
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Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 70-74, 2018 Population and Animal Health, Michigan State Wangchuk K, Wangdi J, Mindu M (2017). University. Available at: Comparison and reliability of techniques to http://www.merckvetmanual.com, (Accessed on estimate live cattle body weight. Short 19 July, 2017). communication. Journal of Applied Animal Upreti CR, Shrestha BK (2006). Nutrient Contents Research. of Feeds and Fodder in Nepal. Animal Nutrition Wangchuk K, Lepcha I, Wangda P (2008). Forage Division, NARC Kathmandu, Nepal. ISBN: Production. A handbook for feed and fodder 99933-703-6-3. development worker in Bhutan. 2nd Edition. RNR RC, Jakar and NFFDP, Bumthang.
Table 3: DMI of DFGs in study districts. Dry Matter Intake of DFGs District Name of DFG Fodder Type Mean Average daily DMI (kg) Total DM% feeding (kg) Dagana Zingchela, Local Fodder 15.76 20 3.15 Tsangkha Improved Pasture 17.74 5 0.89 8.47 Fodder Tree 29.57 15 4.44 Tashiding Local Fodder 15.22 25 3.81 Improved Pasture 19.60 7 1.37 8.96 Fodder Tree 25.22 15 3.78
Tsirang Goserling Local Fodder 27.52 15 4.13 Improved Pasture 22.28 5 1.11 9.35 Fodder Tree 27.42 15 4.11 Patshaling Local Fodder 12.04 20 2.41 Improved Pasture 17.02 5 0.85 8.33 Fodder Tree 25.36 20 5.07
Gasa Damji/Khatoe Local Fodder 9.69 6 0.58 Improved Pasture 17.20 25 4.30 7.78 Fodder Tree 29.03 10 2.90 Yamina/Khailo Local Fodder 13.04 20 2.61 Improved Pasture 17.17 5 0.86 8.44 Fodder Tree 24.89 20 4.98
Punakha Kabjisa Local Fodder 23.32 20 4.66 Improved Pasture 20.28 5 1.01 9.05 Fodder Tree 22.45 15 3.37 Toepaisa Local Fodder 12.72 25 3.18 Improved Pasture 23.96 5 1.20 8.40 Fodder Tree 20.09 20 4.02
Wangdue Busa, Sephu Local Fodder 22.86 15 3.43 Improved Pasture 18.44 15 2.77 8.52 Fodder Tree 46.51 5 2.33 Bajo (Tsedtsho) Local Fodder 29.67 20 5.93 Improved Pasture 25.69 5 1.28 8.73 Fodder Tree 30.16 5 1.51
Mean 8.46 Std. Deviation 0.67
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Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 75-81, 2018
Full length paper Microbiological quality of raw milk in Bhutan
SURYA BC RAI1*, PUSPA M SHARMA2, TENZINLA2, PUNYA MATA2, PEMA TSHOMO2, BHAKTA B GURUNG2
1National Poultry Research and Development Centre, Department of Livestock, Ministry of Agriculture and Forests, Sarpang, Bhutan 2National Centre for Animal Health, Department of Livestock, Ministry of Agriculture and Forests, Serbithang, Thimphu, Bhutan
*Author for correspondence: [email protected]
Copyright © 2018 Surya BC Chamling. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: A two-year longitudinal study was undertaken with the objective to establish a bench-mark for microbiological quality of milk in Bhutan. A total of 2191 milk samples were collected from nine functional and relatively more organized farmers’ dairy groups located in eight different districts and two government dairy farms. Samples were aseptically collected and subjected to rapid field mastitis tests, followed by laboratory culture, isolation, identification and further characterization. The study revealed that 89% of the samples had udder infection, with prevalence rate of sub- clinical and clinical mastitis at 67% and 20.7%, respectively. This indicates contamination of raw milk with both contagious and environmental pathogens. A variety of pathogenic organisms of 18 different groups comprising 944 isolates were isolated and identified. There was a significant prevalence of anti-microbial resistance for milk borne pathogens. There was also a significant seasonal variation in the microbiological quality of raw milk. Based on this study, there is a need to improve the microbiological quality of milk through adoption of hygienic milk production techniques and improved udder health control programme.
Keywords: Raw milk; microbiological quality; mastitis; pathogens; udder health.
1. INTRODUCTION compositional along the whole value chain. Low microbiological content in raw milk prior to The Renewable Natural Resources (RNR) sector processing is essential, as the quality of end aims to achieve self-sufficiency in milk and milk products depends on quality of raw milk. In Kerala, products. The paradigm shift in production system as per the PFA standard, the microbiological from subsistence production system in the past to quality of milk should not exceed 30000 SPC ml-1 commercialization of milk production may and coliform bacteria must be absent in 0.1gm of compromise the microbiological quality of milk. milk (Department of Dairy Development 2010). This shift may have its own detrimental However, in Bhutan, there is no information on consequences in terms of microbiological quality. microbiological quality of raw milk. The faecal Pressures on production system intensification may samples were collected in the morning from the result in production diseases like mastitis. The freshly defecated patch in 30g plastic vials. The emerging concern on human health safety and a fecal sample was analyzed by using three methods rapid growth of dairy industry demand raw milk of of Stoll, sedimentation and floatation, for indicating good quality, both microbiological and 75
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 75-81, 2018 different types of worm prevalence and egg count. or cooperatives, sampling was done at household Thus, this study was undertaken with the objectives level (composite sampling) on the first day, to generate information on microbiological quality followed by individual cow level on the second of raw milk and document important milk borne day, and inspection of cow with sampling on the pathogens in Bhutan, along with antimicrobial third day when required. At the organized susceptibility testing (AST) and influences of other government dairy farms, sampling was done at factors on the microbiological quality of milk. individual cow level on day one, at individual udder level on day-two and revisiting the individual cow with sampling when needed on day-three. 2. MATERIALS AND METHODS Also, at every MPU and government farm, bulk 2.1 Study locations tank milk samples were taken to evaluate the The study locations are presented in Figure 1. Nine quality of bulk tank milk. Every dairy cow at functional farmers’ dairy groups from different farmers’ level and every udder at farm level were districts were purposively selected for the study included in the study. In total, 2191 milk samples (Table 1). The reasons for their selection were were collected and tested. About five milliliters accessibility, existence of functional farmers groups (ml) of milk was aseptically collected in sterile and milk processing units (MPUs). They also bijou bottle with screw-cap as individual sample. represented different agro-ecological zones (AEZs) Samples were coded, stored in cool boxes and of the country. However, the over-riding concern in transported to laboratory. selecting these groups was the public health aspect. These groups produce relatively higher volume of 2.3 Field testing milk and have higher number of consumers for In the field testing, all samples were firstly whom there is safety implication, should the milk subjected to California Mastitis Test (CMT), using quality be unacceptable from microbiological the standard protocol of Ruegg (2005) and aspect. Beside the farmers’ groups, the study was Mellenberger (2001). The individual result obtained also extended to government farms viz. Brown was scored as either N, T, 1, 2 or 3, as per the Swiss Farm in Bumthang district and National interpretation provided in Table 2. In the second Jersey Breeding Centre in Samtse district. The field test, again all samples were subjected to White study comprised of milk sampling and testing at Side Test (WST), as per the modified protocol of field and laboratory at National Centre for Animal Schalm et al. (1971) i.e. 4% NaOH and milk at 3:1 Health (NCAH). ratio. The grading and interpretation of WST were same as that for CMT. The rapid tests were further 2.2 Sampling validated (in the field itself at field veterinary Sampling was done twice in all locations; once laboratories) through somatic cell count (SCC) by each in summer and winter to take into account the breed smear technique. All samples with CMT and possible seasonal variations. In the farmers’ groups
Figure 1: Study locations.
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Table 1: Farmers’ groups and government farms Sl No Farmers groups/Farm District Region Community farm 1 Rama Om Tshogpa Thimphu West 2 Shari Lothuen Om Tshogpa (SLOT) Paro West 3 Samphelling milk Tshogpa Chukha West 4 Choling Yargay Daytshen, Hangay Samtse West 5 Gelephu Milk Detshen Sarpang East-Central 6 Lothuen Om Detshen (LOD) Sarpang East-Central 7 Trong-Dangkhar Gonor Chethuen Tshogpa Zhemgang East-Central 8 Nubi Om Phenden Tshogpa Trongsa East-Central 9 Chokhor Gonor Gongphel Tshogpa Bumthang East-Central Government farm 10 National Jersey Breeding Centre (NJBC) Samtse West 11 Brown Swiss Farm (BSF) Bumthang East-Central
Table 2: Interpretation of CMT scores. by Disk Diffusion Test (Kirby-Bauer method). CMT Score Somatic Interpretation Mueller Hinton Agar plate and antibiotic Cell Range impregnated disc, containing antibiotics as per standards of the Clinical and Laboratory Standards N (Negative) 0 – 200,000 Healthy Quarter Institute (CLSI) were used. A panel of seven T (Trace) 200,000 – Subclinical Mastitis antibiotics commonly used in the field were 400,000 included viz. Ampicillin, Amoxicillin, Tetracycline 1 400,000 – Subclinical Mastitis (broad-spectrum antibiotics), Penicillin G, 1,200,000 Erythromycin (G+ narrow-spectrum antibiotics), 2 1,200,000 – Serious Mastitis Gentamycin and Streptomycin (G- narrow- 5,000,000 Infection spectrum antibiotics). 3 Over Serious Mastitis Result of AST was interpreted on the diameter 5,000,000 Infection of zone of inhibition to nearest mm based on “Zone size interpretation chart” (modified from National Committee for Clinical Laboratory Standards WST scores of 1, 2 and 3 were aseptically (NCCLS) M2 A4: 1990), which is a performance transferred into HiCulture™ Transport Swabs standard for antimicrobial disk susceptibility tests. w/Amies Medium w/Charcoal MS651 for culture, The results were classified into R (resistant), I isolation and identification of bacterial pathogens at (intermediate) and S (susceptible). Moderately NCAH. A few negative (negative to CMT and susceptible class, as per above zone size WST) samples were also included for culture as interpretation chart, was clubbed with intermediate controls to validate the field rapid tests. The FAO’s for convenience. guidelines like CAC/GL 21-1997 and CAC/GL 63- 2007 were also taken into consideration while 2.5. Statistical analysis undertaking this study (FAO 1997 and 2007)4,5. One sample t-test was conducted to test difference CMT scores are directly related to average somatic in pathogen population. The dataset was analysed cell counts. Any reaction of T (trace) or higher with SPSS version 20 and Microsoft Excel. indicates that the quarter has subclinical mastitis. 3. RESULTS 2.4 Laboratory testing 3.1 Important milk borne pathogens/bacterial The samples were finally cultured on Sheep Blood Agar (SBA) and MacConkey Agar at National From the standard bacteriological cultures, 994 Veterinary Laboratory of NCAH. The isolates were major isolates belonging to 18 broad groups were then subjected to various bio-chemical tests (stage obtained (Figure 2). The list included both 1, 2 and 3). Each isolate was sub-cultured to purify, contagious and environmental pathogens. The top multiply and identify the pathogenic organism. ten commonest milk borne pathogens are presented AST was performed for major pathogens isolated in Figure 3.
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35 32.52 30 25 19.17 20 14.83 15 10.59 10 7.52 4.56 4.03 5 1.48 1.38 0.95 0.85 0.85 0.32 0.32 0.21 0.21 0.11 0.11
Isolates (%) Isolates 0
E.coli
Bacillus
Nocardia
Pasteurella
Microcccus
Acinebacter
Enterobacter
Actinobacter
Stahy. aureus Stahy. Enterococcus
Pseudomonas
Streptococcus
Actinobacillus
Proteus vulgaris Proteus
Corynebacterium
Staph. epidermidis Staph. Klebsiella pneumoniae Klebsiella Actinomyces pyogenes Actinomyces
Figure 2: Total milk borne pathogens.
Acti. pyogenes Microcccus 1% Pasteurella 1% Klebsiella pne2%. 4%
Pseudomonas 5% E.coli 33% Streptococcus…
Corynebacterium 11%
Enterobacter Stahy. aureus 20% 15%
Figure 3: Ten commonest milk borne pathogens.
3.2 Antimicrobial Resistance (AMR) of isolates high bovine mastitis prevalence rate of 20.7% for There was a significant difference (prevalence of clinical mastitis and 67% for sub-clinical mastitis. anti-microbial resistance) between sample mean Among the results obtained, this is one of the most and hypothesized mean, indicating presence of anti- important baseline information generated. microbial resistance in milk borne pathogens. The AST profiles are given in Table 3, 4 and Figure 4. 4. DISCUSSION
The most common mastitis causing organism was 3.1 Seasonal variation and mastitis prevalence found to be Escherichia coli (E. coli), followed by There was a significant seasonal variation of Staphylococcus aureus and Streptococcus. This is mastitis with a higher prevalence in winter (Figure similar to the findings of Bradley (2001), 5). Compared with serious mastitis, the prevalence Miltenburg (1996), Schukken (1989), Burvenich of sub-clinical mastitis was significantly higher in (2003), and Petrovski (2006). Presence of both both summer and winter. This study revealed a very Rai et al. (2018) 78
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 75-81, 2018
60 R I S
50 48 42 42 39 40 36 32 30 24
20 17 14 14 11 12 11 9 10 9 10 4 4 2 3 2 0 Amx ERY PEN G Gen Amp Tet Strep
Figure 4: AST profile.
Table 3: AST by most sensitive and most 80 Sub-clinical Mastitis resistant. 70 Serious Mastitis Most sensitive Most resistant 60 Tetracycline 27%, Pen G 23% 50 Streptomycin 17%, Ampicillin 20%
% Amoxicillin 17% Amoxicillin 20% 40
30 Table 4: AST profile (S=Susceptible, 20 I=Intermediate, R=Resistant). % 10 Isolate S I R 0 Summer Winter Penicillin G 6.00 3.00 23.0 Gentamycin 3.00 34.0 7.00 Figure 5: Seasonal mastitis prevalence. Tetracycline 27.0 21.0 7.00 Streptomycin 17.0 28.0 6.00 contagious and environmental pathogens indicates Amoxicillin 17.0 2.00 20.0 pathogenic organisms having entrenched in our Erythromycin 16.0 8.00 17.0 dairy herds. The environmental factors related to Ampicillin 14.0 4.00 20.0 poor management, particularly the housing part contribute to high microbial load. Since there is no strict legislation or guideline encourage prudent use of antimicrobials as put for anti-microbial usage for mastitis treatment and forward by Oliver et al. (2012). A study at control, some levels of antibiotic resistance are Michigan State University by Erskine (2002) expected in such a scenario. Resistance proportion showed that there is no indication of increased ranging from 23% for penicillin G to 6% for resistance of mastitis isolates to antibacterial that Streptomycin is comparable with the findings of are commonly used in dairy cattle. This was made Saini et al. (2012). The high proportion of antibiotic possible through prudent use of antimicrobials resistance for penicillin G was also found by Botrel aided by strict legislation on the use of et.al (2010) who estimated the distribution of antimicrobials. pathogens, as well as their antimicrobial resistance Bacterial antimicrobial resistance in both pattern, in cows affected by clinical or subclinical medical and agricultural fields has become a mastitis in the Rhône-Alpes region of France. Thus, serious concern worldwide. Research has linked the this study stipulates the need for strategies to use of antibiotics in agriculture to the emergence of Rai et al. (2018) 79
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 75-81, 2018 antibiotic-resistant food borne pathogens E. coli mastitis is mainly determined by cow (McDermott et al. 2002). factors. Veterinary Research, 34 (5): 521-64. Although the seasonal effect on prevalence of Botrel MA, Haenni M, Morignat E, Sulpice mastitis was unexpected, we found that the P, Madec JY and Calavas D (2010). prevalence was significantly higher in winter. This Distribution and antimicrobial resistance of is in agreement with the finding of highest clinical clinical and subclinical mastitis pathogens in mastitis incidence rate in December to January dairy cows in Rhône-Alpes, France. Foodborne (Olde Riekerink et al. 2007). The higher winter Pathogens and Disease, 7 (5): 479-87. prevalence could be attributed to poor personal and Bradley AJ and Green MJ (2001). Aetiology of equipment hygiene under the freezing cold in clinical mastitis in six Somerset dairy herds. winter. Veterinary Record, 148 (22): 683-6. Even though, a high prevalence rate was Department of Dairy Development (2010). detected, it is comparable to existing prevalence Government of Kerala. Available at: rates in Asia (Sharma et al. 2012) who showed an http://www.dairy.kerala.gov.in/index.php/qualit increasing trend of bovine mastitis with a y-control/pfa-standards-of-milk. (Accessed on prevalence of >70% for India, >60% for Pakistan 19th March 2018) and Nepal, >50% for Bangladesh, South Korea and Erskine RJ, Walker RD, Bolin CA, Bartlett PC and China. However, given the free veterinary services White DG (2002). Trends in antibacterial provided by the government in Bhutan, it can be susceptibility of mastitis pathogens during a brought down significantly with an improved udder seven-year period. Journal of Dairy Science, 85 management and addressing the other risk factors (5): 1111-8. of bovine mastitis. FAO (2007). Principles and guidelines for theconduct of microbiological risk management 5. CONCLUSION (MRM) CAC/GL 63-2007. Available at: This study revealed a large number of bacterial file:///C:/Users/user/Downloads/CXG_063e.pdf pathogens responsible for bovine mastitis. The , (Accessed on December 3, 2016). definite prevalence of anti-microbial resistance too McDermott PF, Zhao S, Wagner DD, Simjee was proven. A high prevalence rate of bovine S, Walker RD and White DG (2002). The food mastitis with higher prevalence in winter season safety perspective of antibiotic resistance. was established. To address the issue of poor Animal Biotechnology, 13 (1): 71-84. microbiological quality of milk, a bovine udder Mellenberger R (2001). California Mastitis Test health control program may be instituted in near (CMT)- An Invaluable Tool for Managing future. Although, this study provided lot of Mastitis. Dept. of Animal Sciences, Michigan information on microbiological quality of raw milk, State University. Available at: there is a need to address the gaps in this study and http://immucell.com/wp-content/uploads/An- establish more valid benchmarks. Some of the areas Invaluable-Tool.pdf. (Accessed on 20 February to focus in future studies are correlation with other 2018) host factors viz., breed, lactation number, lactation Miltenburg JD, de Lange D, Crauwels AP, Bongers stage, age, parity, length of dry period, milking JH, Tielen MJ, Schukken YH and Elbers AR interval and environmental factors like housing, (1996). Incidence of clinical mastitis in a nutrition, hygiene, milking techniques etc. Besides random sample of dairy herds in the southern these, improvement in the overall study design, Netherlands. Veterinary Record, 139 (9): 204- including laboratory protocols, is crucial. 7. Olde Riekerink RG, Barkema HW and Stryhn H Acknowledgements (2007). The effect of season on somatic cell The authors would like to express their appreciation count and the incidence of clinical mastitis, 90 to all district extension staff, technicians in various (4): 1704-15. veterinary laboratories, staff in government farms Oliver SP and Murinda SE (2012). Antimicrobial and employees of different farmers’ milk resistance of mastitis pathogens. Veterinary cooperatives, without whose pro-active support, Clinics of North America, Food Animal this research would not have materialized. Practice, 28 (2):165-85. Petrovski KR, Williamson NB, Lopez-Villalobos REFERENCES N, Parkinson TJ, and Tucker IG (2006). Culture results from milk samples submitted to Burvenich C, Van Merris V, Mehrzad J, Diez- veterinary diagnostic laboratories from August Fraile A and Duchateau L (2003). Severity of 2003 to December 2006 in New Zealand. New
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Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 75-81, 2018 Zealand Veterinary Journal 2011 Schukken YH, Kremer WD and Lohuis JA (1989). Nov;59(6):317-22. Escherichia coli mastitis in cattle. I. Clinical Ruegg PL (2005). California Mastitis Test (CMT) diagnosis and epidemiological aspects. Tijdschr Fact Sheet 1 & 2. Available at: Diergeneeskd, 114 (15-16): 829-38. http://milkquality.wisc.edu/wp- Sharma N, Rho GJ, Ho Hong Y, Kang TY, Lee content/uploads/2011/09/california-mastitis- HK, Hur TY and Jeong DK (2012). Bovine test-fact-sheet.pdf, Mastitis: An Asian Perspective. Asian Journal http://milkquality.wisc.edu/wp- of Animal and Veterinary Advances, 7: 454- content/uploads/2011/09/california-mastitis- 476. test-fact-sheet-2.pdf. (Accessed on 23 February 2018). Saini V, McClure JT, Léger D, Keefe GP, Scholl DT, Morck DW, and Barkema HW (2012). Antimicrobial resistance profiles of common mastitis pathogens on Canadian dairy farms. Journal of Dairy Science, 95 (8): 4319-32.
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Full length paper Foot and Mouth Disease (FMD): Outbreak containment at cattle herd level in Samdrup Jongkhar district
LUNGTEN LUNGTEN1*, TENZIN TENZIN2, KAROON CHANACHAI3 AND TSHEWANG RABGAY1
1Satellite Veterinary Laboratory, Department of Livestock, Ministry of Agriculture and Forests, Deothang, Samdrup Jongkhar, Bhutan 2National Centre for Animal Health, Department of Livestock, Ministry of Agriculture and Forests, Thimphu, Bhutan 3Bureau of Disease Control and Veterinary Services, Department of Livestock Development, Bangkok, Thailand
*Author for correspondence: [email protected]
Copyright © 2018 Lungten Lungten. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: A study was conducted with the objective to determine the risk factors of Foot and Mouth Disease (FMD) outbreak occurrence in Samdrup Jongkhar district in Bhutan. Twenty-six household that had experienced FMD outbreak and diagnosed as FMD in cattle between January 2012 and December 2015 were defined as a case. Two households that owned cattle and had not experienced any outbreaks of FMD within the same village were selected as control. Data related to management and environmental factors were collected with the questionnaire survey during November and December 2016. The univariate and multivariate logistic regression models were built to identify risk factors of FMD. The adjusted odds of FMD outbreak occurrence was 3.91 times greater in the households that had large herd size (>5 cattle) when compared to small herd size (<5 cattle). Similarly, the occurrence of FMD was significantly higher in herds that had not vaccinated against FMD than those vaccinated. Therefore, the FMD control strategy such as bi-annual vaccination is still an effective tool to prevent FMD outbreak. The findings recommend further study to explore why large herd size has higher chance of FMD infection.
Keywords: Case control study; cattle; Foot and Mouth Disease; herd; risk factors.
1. INTRODUCTION losses. FMD is endemic in Bhutan but most commonly prevalent in southern districts bordering Foot and Mouth Disease (FMD) is a highly India (Dukpa et al. 2011a). An average of 18 contagious viral disease that affects all cloven- outbreaks occur annually in the country. Serotype O footed animals. It is characterized by pyrexia, is involved in FMD outbreaks in Bhutan (Dukpa et salivations, vesicles formation on mouth and buccal al. 2011a). Bhutan practices annual and also bi- cavity, interdigital space, and teats, causing annual mass vaccination against FMD with a target occasional death in young animals. The disease is to achieve at least Stage 3 of the FAO/OIE endemic in many parts of the world, particularly in Progressive Control Pathway by 2020 (NCAH developing countries of Asia, Africa, Middle East, 2016). Previous study conducted in the central and and some parts of Europe, leading to huge economic western part of the country had identified several 82
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 82-86, 2018 risk factors (e.g. management and animal level workers and the case was further investigated by factors) for the occurrence of FMD outbreaks Veterinary Officials of District Veterinary Hospital (Dukpa et al. 2011b), but there is only scarce and Satellite Veterinary Laboratory. The FMD cases information about disease epidemiology in south- in animals were clinically diagnosed in the field and eastern region of the country. In order to develop also confirmed at the laboratory. Further, the prevention and control strategy, information about epithelial tissue samples were collected and referred the disease epidemiology is essential. Therefore, a to the international laboratory for molecular study was conducted with the objective to determine analysis. The location of each case household was the risk factors of FMD outbreak occurrence, using retrieved from the database maintained at the the retrospective case-control study design. District Veterinary hospital, Satellite Veterinary Laboratory and online transboundary animal disease 2. MATERIALS AND METHOD information system (TADInfo). The database was further verified during field visits with the help of 2.1 Study area the sub-district control household in the same The study was conducted in Samdrup Jongkhar village. A household from another village nearer to district, which is located in south-eastern Bhutan and the case village was selected. The information about shares border with India (Figure 1). The district has the control households that have not experienced any 11 sub-districts and each sub-district livestock cases of FMD in their cattle during the study period centre has livestock development workers who were verified with the help of sub-district livestock provide veterinary and production related services to development workers. Seventy-eight households (26 the community. Nine villages of seven sub-districts case and 52 control households) from nine villages were selected for the study. and seven sub-districts were selected and interviewed (Table 1). 2.2 Study design A retrospective case-control study design was used 2.3 Data collection and analysis to collect data at the herd level. Within a village, A structured questionnaire with closed questions households or herds that had experienced FMD was used for the study. The questionnaire was outbreak in cattle during January 2011 to December pretested with five farmers prior to actual survey. 2015 were selected as case. Two cattle owning Owners of each case were interviewed. The households within the same village that have not geographic coordinates of each case and control experienced any outbreaks of FMD were randomly household were collected. The data were entered selected as controls. FMD outbreaks were initially into a database developed in EpiInfoTM version investigated and reported by livestock development 7.1.2.0 (Centers for Disease Control and Prevention
Figure 1: Study areas in Samdrup Jongkhar district.
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Table 1: Locations and number of households final multivariable model, two variables were found selected and interviewed. to be associated with FMD outbreak occurrence. The odds of FMD outbreak occurrence was about four Number of households times greater in the households that had large herd Villages Case Control Total size (>5 cattle) when compared with small herd size (<5 cattle). Significantly higher odds of FMD Domphu 5 10 15 occurrence was observed in herds that were not Khenorong 1 2 3 vaccinated against FMD than those vaccinated Richanglu 4 8 12 (Table 3). The model diagnostic test revealed best fit Jangsa 4 8 12 of the model without any outliers (Dahoo et al. 2009). Durtsi 1 2 3
Samrang 1 2 3 4. DISCUSSION Tshothang 8 16 24 We determined the risk factors associated with Zangthi 2 4 6 outbreaks of FMD in cattle at the household level in Total 26 52 78 Samdrup Jongkhar district, using a case-control study design. Except for one outbreak that occurred during winter (December), all other outbreaks (CDC), Atlanta, GA, USA) and analyzed using (n=25) in the study area occurred during summer STATA version 14.0 (StataCorp USA). season (June-August), which is a normal trend in In order to understand the risk factors of FMD Bhutan (Dukpa et al. 2011a; Tenzin and Thapa occurrence at the household level, an unconditional 2016). Summer season in Samdrup Jongkar is hot univariate logistic regression analysis was and humid, which causes stress to the animals and performed. The model used FMD status (case vs may predispose them to infections (Dohms and Metz control households) as the outcome and each risk 1991). indicator variable as predictor variable. Predictor Herd size was an important risk factor for the variables associated with FMD outbreak status with occurrence of FMD in the study area, indicating that p value less than 0.25 were selected for multivariate the probability of experiencing FMD outbreak logistic regression analysis. A forward stepwise increases with increase in herd size (i.e. the more method of mixed-effect multiple logistic regression animals that are at risk, the greater the likelihood that analysis was performed to identify risk factors for at least one of them will acquire infection). Similar FMD occurrence. To determine the best fitting findings have been reported in a previous study in model, a variable with the smallest p-value based on Bhutan (Dukpa et al. 2011a) and elsewhere in the log likelihood ratio statistics in the univariate world (Khounsy et al. 2008; Verma et al. 2008) analysis was entered into the model first. Thereafter, where a higher animal density was found to be each of the remaining variable was added to the associated with an increased FMD incidences. In the model containing the first variable to test whether its village context in Bhutan, farmers that own smaller addition significantly improved the fit of the model. number of animals mostly keep their animals within The variable with the highest likelihood ratio the cattle shed or by tethering in the nearby field or statistic (chi-square test with one degree of freedom) household compound and are managed by stall was selected for addition to the model and the feeding. Thus, there may be limited interaction with process was then repeated. The final model was other animals in the village. In contrast, those selected based on the significant p value (p≤0.05). households that have large number of cattle are often Following the fitting of the main effects model, the let out in the forests for grazing due to fodder goodness of fit of the model was assessed by shortage and other management difficulties. examining the residuals and outliers. These animals come into contact with other
households’ animals, including wildlife at the 3. RESULTS common grazing ground and water source, thus The respondents comprised of 48 (61.54%) males posing risk of contracting infectious diseases, and 30 (38.46%) females with a mean age of 47 including FMDV. Therefore, it is important to create years. Each respondent’s household owned about awareness among farmers on the risk of contracting seven cattle at the time of outbreak. In the study infectious diseases during open grazing and options areas, the outbreak occurred mostly in summer and for limiting disease outbreaks through improved winter seasons. pasture development. Ten variables were identified as risk factors of This study also demonstrates that an annual FMD outbreak occurrence at the household level FMD vaccination in animals decreased the odds of (Table 2). When adjusted for other variables in the
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Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 82-86, 2018 FMD occurrence in cattle, compared with those vaccines are distributed free of charge by the herds that have not been vaccinated regularly. government, several logistical challenges in the Large scale outbreaks of FMD in cattle have remote villages result in low coverage of been reported previously in non-vaccinated herds in vaccination. For instance, there is only one livestock central Bhutan (Tenzin 2007) and also during recent extension staff in each sub-district that looks after outbreak in Paro in western Bhutan. Although, FMD animal health (preventive and curative). Production
Table 2: Univariate logistic regression analysis of the risk factors for the occurrence of FMD outbreak at the household level, Samdrup Jongkhar district. Variables and categories b SE OR (95% CI) p-value i. Cattle herd size (No of cattle) <5 cattle 0 1 >5 cattle 1.469 0.51 4.34(1.60-11.80) 0.004 ii. Presence of bulls/bullocks in the herd during the time of outbreak No 0 1 Yes 0.713 0.497 2.04(0.77-5.40) 0.151 iii. Keeping animals in open space (outside shed) No 0 1 Yes 1.006 0.522 2.73(0.98-7.61) 0.054 iv. Grazing of cattle in the forests No 0 1 Yes 0.749 0.498 2.11(0.80-5.62) 0.133 v. Mixing of cattle with animals of other owners or village No 0 1 Yes 1.567 0.677 4.80(1.27-18.05) 0.021 vi. Drinking water source for cattle Tap water 0 1 Open source/stream 1.485 0.678 4.41(1.17-16.67) 0.029 vii. Mixing of cattle with other cattle at the water source No 0 1 Yes 1.485 0.678 4.41(1.17-16.67) 0.029 viii. Hiring out of the bulls/bullocks by the household for ploughing No 0 1 Yes 0.706 0.488 2.03(0.78-5.27) 0.147 ix. Letting out of the bulls/bullocks in the forest for free grazing No 0 1 Yes 0.999 0.502 2.71(1.02-7.25) 0.047 x. Whether the HH practice seasonal migration of cattle No 0 1 Yes 1.258 0.54 3.50(1.21-10.10) 0.02 xi. FMD vaccination status of cattle in the herd Vaccinated 0 1 Not vaccinated 2.094 0.613 8.12 (2.44-26.97) 0.001
Table 3: Final multivariable logistic regression model for FMD outbreak occurrence at the household level in Samdrup Jongkhar district. Variables and categories b SE OR(95%CI) P-Value
Constant -2.594 0.617 - - Cattle herd size <5 cattle 0 1 >5 cattle 1.364 0.557 3.91 (1.31-11.67) 0.012 FMD vaccination status of cattle in the herd Vaccinated 0 1 Not vaccinated 2.015 0.634 7.50 (2.16-26.03) 0.0004
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Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 82-86, 2018 and extension work are carried out in a scattered and Bronsvoort BM, Nfon C, Hamman SM, Tanya remote community, which makes the task of VN, Kitching RP and Morgan KL (2004). Risk vaccinating thousands of animals difficult and leads factors for herdsman-reported foot-and-mouth to low vaccination coverage. In addition, the cattle disease in the Adamawa Province of are usually let out for grazing in the forests when Cameroon. Preventive Veterinary Medicine, 66 extension officials visit the household for (1-4): 127-139. vaccination, leading to non-vaccination of animals, Cleland PC, Baldock FC, Chamnanpood P and thus, becoming susceptible to infection. Further, Gleeson LJ (1996). Village level risk factors for some owners may not allow their animals to be foot-and-mouth disease in northern Thailand. vaccinated against FMD believing that vaccination Preventive Veterinary Medicine, 26 (3-4): 253- would result in abortion of pregnant animals and 261. reduction in milk yield. Therefore, it is important to Dohms JE and Metz A (1991). Stress mechanisms of create awareness among farmers on cost-benefit of immunosuppression. Veterinary Immunology vaccination versus economic loss due to infection. and Immunopathology, 30 (1): 89-109. Since Samdrup Jongkhar district is classified as Dohoo I, Martin W and Stryhn H (2009). Veterinary high-risk zone for FMD, a bi-annual vaccination Epidemiologic Research. AVC Inc., program with more than 80% coverage is required to Charlottetown, Canada. prevent outbreaks, as per the National FMD Dukpa K, Robertson ID, Edwards JR and Ellis TM Prevention and Control Plan 2016 (NCAH 2016). (2011a). A retrospective study on the The relatively small sample size may have epidemiology of foot-and-mouth disease in affected the ability of risk factors to be retained in Bhutan. Tropical Animal Health and the final model. Most of these characteristics were Production, 43: 495-502. found to be significant risk factors for FMD Dukpa K, Robertson ID, Edwards JR, Ellis TM, occurrence in Bhutan in the previous study (Dukpa Tshering P, Rinzin K and Dahal N (2011b). Risk et al. 2011b) and elsewhere in the world (Cleland et factors for foot-and-mouth disease in sedentary al. 1996; Bronsvoort et al. 2004). livestock herds in selected villages in four The main limitation of this study is small sample regions of Bhutan. New Zealand Veterinary size where only 26 cases and 52 control households Journal, 59 (2): 51-58. were interviewed, which is demonstrated by wide Khounsy S, Conlan JV, Gleeson LJ, Westbury HA, confidence interval of the odd ratio. The Colling A, Paton DJ, Knowles NJ, Ferris NR and retrospective nature of case-control study design in Blacksell SD (2008). Foot and mouth disease in this study could have also affected the findings due the Lao People's Democratic Republic: I. A to recall bias. Nevertheless, it has provided review of recent outbreaks and lessons from important information to guide the policy decision control programmes. Review of Scientific on FMD prevention and control options in Samdrup Technology, 27 (3): 839-849. Jongkhar and elsewhere in the country. National Centre for Animal Health (2016). National Foot and mouth disease Prevention and Control 5. CONCLUSION Plan. 3rd Edition, National Centre for Animal Health, Department of Livestock, Thimphu, This study has identified herd size and management Bhutan. risk factors to be targeted for prevention and control Tenzin T (2007). Foot and Mouth Disease outbreaks of FMD and future research. Promoting a bi-annual at Shingkhar, Bardo, Nangkhor and Trong geogs vaccination protocol as per the National FMD under ZhemgangDzongkhag: Investigation Prevention and Control Plan for cattle against FMD report. National Centre for Animal Health should build up herd immunity and prevent Serbithang, Thimphu, Bhutan. outbreaks. Similarly, cattle management system Tenzin T and Thapa L (2016). Status of notifiable including biosecurity should be improved to prevent animal disease in Bhutan-1996-2016. National animals from acquiring infectious diseases. Centre for Animal Health, Serbithang, Bhutan.
Verma AK, Pal BC, Singh CP, Udit J, Yadav SK Acknowledgements and Mahima (2008). Studies of the outbreaks of We acknowledge the support provided by Regional foot and mouth disease in Uttar Pradesh, India, Livestock Development Centre, Kanglung and the between 2000 and 2006. Asian Journal of Livestock Extension Officials of Samdrup Jongkhar Epidemiology, 1: 40–46. district on data collection. The fund support of FAO-
RAP Bangkok is acknowledged, which enabled the first author to participate in the FETP-V course.
REFERENCES
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Full length paper Plant extracts as alternatives to anti-anthelmintic drugs: findings from Maedwang subdistrict, Thimphu
NIDUP DORJI* AND PHUB DORJI
Department of Animal Science, College of Natural Resources, Royal University of Bhutan, Lobesa, Punakha
*Author for correspondence: [email protected]
Copyright © 2018 Nidup Dorji. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: A study was conducted to investigate the prevalence of endo-parasites in dairy cattle and evaluate anti-anthelmintic properties of common wormwood (Artemisia vulgaris) leaves, papaya (Carica papaya) seed and mustard (Brassica nigra) oil. The plants were collected, sun dried, pulverized and mixed with warm water to prepare plant suspensions. The suspensions were administered to 3 groups of cattle, with each group consisting 10 cattle. Out of 104 fecal samples analyzed, about 65.38% were positive, including Strongyles 2.88%, Ascaris 39.42%, Coccidia 11.53%, Fasciola 0.96% and mixed infection 10.57%. All plants suspensions were effective in reducing the egg count but mustard oil proved to be more effective. The study concluded that all three plant suspensions were effective against endo-parasite and have potentials to enhance milk production.
Keywords: Anti-anthelmintic; common wormwood; mustard oil; papaya seed; plant suspension.
1. INTRODUCTION livestock. Due to parasitic problem, high numbers of synthetic de-worming drugs are being used incurring Agriculture is the backbone of rural economy. Over huge budget expenditure. Similarly, in Bhutan, a 62% of Bhutanese population are engaged in huge amount of budget is spent on procurement and farming activities (PPD 2013). Farmers rear usage of synthetic deworming drugs. As per the livestock of different breeds and livestock record of National Center for Animal Health, contributes about 3.79% to the total Gross Domestic Serbithang, there has been an expenditure of over Product (DoL 2012). Livestock provides a sense of Nu. 8 million in three consecutive years from 2012 security to rural farmers in times of crop failure since to 2014. With high usage of deworming drugs, issues they can be exchanged readily for cash or food grain. of drugs resistance are being reported (Manthri et al. Livestock, especially dairy cattle are reared under 2011). It calls for alternative methods to address the semi-extensive systems where they graze freely in issue. One of the alternatives could be the use of forest and community grazing land. This system plant products. Some plants are reported to have exposes cattle to parasites and other communicable potential to combat the prevalence of parasite in diseases. Terbalanche (1979) attributed the problem cattle since it has anti-anthelmintic properties. There to more number of parasitic cases emerging due to are evidences from India, South Africa and China on poor pasture grazing management system, the use of these plants as alternate to synthetic anti- hampering production capabilities and health of anthelmintic drugs (Ameen et al. 2010). 87
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In Bhutan, there is traditional practice of using of improved cattle (21,891 female cattle) (DoL plants to treat diseases. However, there is lack of 2013). scientific evidence on the use of certain plants for 2.2 Experimental design and treatment treatment of diseases. Therefore, a study was The experiment was conducted using the conducted with the main objectives to investigate the Completely Randomized Design (CRD). The prevalence of endo-parasites infestation in cattle and experimental treatments were plant suspensions of evaluate the efficacy and anti-anthelmintic common wormwood (Artemisia vulgaris) leaves properties of Artemisia leaf and papaya seed in the (T1), papaya (Carica papaya) seed (T2) and mustard treatment of endo-parasites. These plants could be an (Brassica nigra) oil (T3). The Draw Lots method alternative to synthetic drugs and minimize the use was used for sampling 66 infected cattle. Of these, of synthetic deworming drugs. 38 cattle had no infestation. Only 30 endo-parasite infected dairy cattle were randomly selected and tied 2. MATERIALS AND METHOD in one place for identification of dung patches. The cattle were divided into three groups with each group 2.1 Study site consisting 10 cattle. The plant suspensions were Maedwang subdistrict under Thimphu district was administered to the groups. selected for the study (Figure 1). The subdistrict was selected because it had the highest number of endo- 2.3 Cattle sampling and dosage administration parasitic cases among subdistricts, as per the clinical The animals were administered with suspensions of records of District Veterinary Hospital. The study common wormwood leaves, papaya seed, and was conducted in Tsalluna under Maedwang mustard oil. The sample with initial egg count was subdistrict located in the south-east of Thimphu used as control. Dosage for each herbal plant extract (Figure 1). Tsalluna comprises of 17 villages with an was administered at 200 g in 500 ml of warm water area of 223.24 sq. km (NFYP 2012). The subdistrict per adult cattle and mustard oil was administered at is enriched with fertile soils that are good for 200 ml per cattle. Fecal samples from treated agriculture farming. Tsalluna was selected because animals were collected after every one week for egg it has large number of dairy groups and cattle count assessment. The samples were collected in 30g population. The subdistrict has about 22,149 cattle plastic vials in the morning from the freshly heads that include 208 local cattle with more number defecated patch. The samples were analyzed using of female cattle (145 numbers) and 21,941 numbers stoll, sedimentation and floatation methods, to detect different types of worm prevalence and egg count.
Figure 1: Map and location of study area.
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3500 3328 About 65% samples tested positive to endo-parasites comprising 39.42% Ascaris, 11.53% Coccidia, 3000 2.88% Strongyles and 0.96% fasciola (Figure 2). About 10.57% were mixed infection and 34.62% 2500 tested negative.
2000 3.2 Effects of plant suspension on endo-parasites The effects of plant suspension on endo-parasites at
No. of samples of No. 1500 different intervals are presented in Table 1. For the wormwood treatment, the initial mean egg count was 8200 eggs per gram (epg) and after treatment, the 1000 count was 5100, 2100, 1400 epg on week one, two 521 453 and three, respectively. The mean egg count was 500 372 277 207 153 reduced by 83% in three weeks. There was a 78 significant difference in worm load (epg counts) 0 among four treatment intervals. BV EQ PR OV CP CA AV OT For the papaya seed treatment, before treatment, Animal species the worm count was 8700 and the worm counts were 2300, 2800 and 1700 epg on week one, two and Figure 2: Prevalence of endo-parasites. three, respectively. There was a significant difference in worm load (egg counts) between
treatment intervals. This indicates that papaya seeds 2.4 Preparation of plant suspension have de-worming properties and could control endo- The dry wormwood leaves were collected in the parasites such as Ascaris and Coccidia. However, month of December, 2014 from Jhazam area under there was no statistical difference between first, Thimphu. According to National Institute of second and third treatment, which means repeated Traditional Medicine (NITM), the abundantly dose after first treatment has no concurrent effect grown species of Artemisia is Vulgaris species. The unlike wormwood leaves suspension. ripped papaya was collected from Gelephu and Under treatment with mustard oil, the worm Phuntsholing. The seeds were extracted from papaya count was 6400 epg before treatment, and in the pods and washed with tap water and sun dried for 3 entire treatment course, the worm count in week one days until seeds have dried completely. The was 2400 epg, week two 1700 epg and week three wormwood leaves and papaya seeds were pulverized 900 epg. The significant difference was observed in in an electric grinder for 15 minutes and sieved for epg count with mustard oil treatment at different the final powder. The powder was weighed to 200 g intervals. The results obtained before and after and blended with 500 ml of warm water. The treatment (one week, two weeks and three weeks suspension was kept overnight and then interval) were significantly different. This indicates administered in the next day. After treatment, the that mustard oil does have anthelmintic effects. fecal samples were collected for egg counts. Mustard However, repeated treatments did not show much oil was purchased directly from the market and efficacy as compared to other two treatments. administered at 200 ml per treatment to large animal and around 100 ml to young cattle. A total of 104 4. DISCUSSION fecal samples were collected and analyzed. Out of 104 samples analyzed, the test results show 2.5 Data analysis high prevalence of parasites in the study area. This Primary data were collected by using semi- agrees with the finding of National Center for structured questionnaire. Graphs were prepared in Animal Health (NCAH 2013). The study area has Microsoft Excel. An analysis of variance and a open grazing land, which seems to have been friedman’s test (mean and standard deviation) were contaminated by parasite. The infected cattle also conducted to determine reduction in endo-parasitic appear to have developed resistance due to frequent load under three different treatments. Pairwise usage of drugs. Resistance of internal parasite of comparisons were made to detect significant livestock to drugs is reported worldwide (Waller et differences between treatments. The dataset was al. 2001). analyzed with SPSS software version 16.0. Most wormwood species have de-worming properties (Laudato and Capass 2013). Junquera 3. RESULTS (2014) reported that the whole plant of wormwood has constituents of “Thujone”, which is used against 3.1 Composition of infection Dorji and Dorji (2018) 89
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Table 1: Mean epg count under three plants suspension treatments at different intervals (Mean SD). Means with different letters within a row differ significantly at p≤0.05. Count 1 week Reductio Before 2 weeks after 3 weeks after Treatment after n in final worm treatment treatment treatment treatment count (%)
Wormwood plant extract 820399 a 510292 b 210129 c 14070 d 83.0 Papaya plant extract 870523 a 230221 b 280235 b 17082 b 80.0 Mustard plant oil 640532 a 240375 b 170125 b 9032 b 86.0
parasitic round worms (e.g. Haemonchus, This study tested plant extracts in cattle only. There Bunostomum and Protostrongylus). Repeated dose is a need to extend such studies to other animal of plant extract at different intervals helped in species such as equine, swine and canine. eliminating worms. This could be due to consistent use of oil in the stipulated time intervals. Debairacli Acknowledgements (1973) reported that the infusion of dry flower of The authors would like to thank District Livestock wormwood is noxious for Bunostomum, Sector, Thimphu, for financial assistance. We are Dictyocaulus and Protostrongylus. Lans et al. (2007) also thankful to Dr. Phuntsho Wangdi, confirmed that the de-worming of pets and pigs with Parasitologist, NCAH, Mrs. Sangay Wangmo, and A. absinthium, A. annua, A. cina and A. vulgaris was Mr. Karma Wangdi, Laboratory Technician and Mr. effective against Ascaris. Elgarhy and Mahmoud Partiman Rai, LEC, Tsalluna, for rendering (2002) also found that wormwood seeds effectively assistance in field survey and collection of fecal reduced the egg count of Strongyloides. samples. The authors are also grateful to farmers of Repeated treatment with papaya seed Maedwang subdistrict for providing their animals suspension showed no concurrent effects on for the study. parasites. This might be due to less concentrations of plant extract resulting in less effect on worms. It REFERENCES contradicts the finding of Satrija et al. (1994) who Ameen SA, Adedeji OS, Ojedapo LO, Salihu and reported that the use of water extracts of papaya Fabusuyin CO (2010). Anthelmintic potency of seeds decreases Ascaridia galli infections in poultry. papaw (carica papaya) seeds in Western African The papain in papaya seed has been reported to Dwarf (WAD) sheep. Department of Animal reduce worms (Ameen et al. 2010). The main Production and Health, Ladoke Akintola anthelmintic property in papaya is benzyl University of Technology, P.M.B. 4000, isothiocyanate that destroys worms (Krishna et al. Ogbomoso, Nigeria, Natural Medicine 2008). In a study conducted by Ameen et al. (2010), Development Agency, V.I. Lagos. the powder and aqueous solution treatment Coles GC (1995). Chemotherapy of human significantly reduced worm burden in sheep. nematodes: learning from the problems in sheep. Mustard oil proved effective during initial Journal of the Royal Society of Medicine, 88: treatments but after repeated treatment it became 649–651. less effective. This may be due to less dosage Debairacli LJ (1973). Herbal handbook for farm and (200ml) per adult cattle. The oil has anthelmintic stable, Faber and Faber, London. ingredients, which was reported to cause death of DoA (2012). Agriculture Statistics. Ministry of earthworms (Manthri et al. 2001; Manthri et al. Agriculture. 2011). DoL (2012). Livestock Statistics. Ministry of
Agriculture and Forest, Thimphu: Bhutan. 5. CONCLUSIONS DoL (2013). Livestock statistics. Ministry of Plants extracts and oil tested in this study proved to Agriculture and Forest, Thimphu, Bhutan. be effective in controlling worms. Similar study Duval J (1994). Christiane Trudeau of the McGill needs to be carried out to test the effectiveness of University Institute of Parasitology, Jean-Marie other vegetable extracts of pumpkin seed, ginger, Boucher of the Centre d'Agriculture Biologique garlic, marigold flower and onion. In this study, the de la Pocatièr. plant suspensions were prepared with water. Elgarhy MF and Mahmoud LH (2002). Enthelmintic However, the use of these plants with other medium efficacy of traditional herbs on Ascaris and extracts should be explored in future studies.
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lumbricoides. Journal of Egyptian Society for PPD (2013). Renwable Natural Resources Census. Parasitology, 32(3): 893-900. Ministry Agriculture and Forestry, Royal Ferriran JFS (undated). Artemisia Species in Small Government of Bhutan, Thimphu. Ruminant Production: their potential Satrija F, Nansen S and Murtini SH (1995). Antioxidant and Anthelmintic Effects. Research Anthelmintic activity of papaya latex against Horticulture, USDA, ARS, Appalachian patent Heligmosomoides polygyrus infections Farming System Research Centre, 1224 Airpot in mice. Journal of Ethnopharmacology, 48 (3): Rd., Beaver, WV 25801. 161-164. Junquera P (2014). Roundworms: parasitic worms Terbalanche EJ (1979). Trials with febental to of dogs, cats, cattle, sheep, pigs and poultry. determine the affections breeding ewes and their nematodes, helminthes. offsprings. Veterinary Medicine Review, 79: Krishna KL, Paridhavi M and Patel JA (2008). 118-125. Review on nutritional, medicinal and Villarroel A (2013). Internal parasites in sheep and pharmacological properties of Papaya (Carrica goats, Oregon State University, the U.S. papaya Linn). JSS Collegeof Pharmancy, SS Department of Agriculture, and in Oregon Nagara, Mysore-570 015, Karnataka, India. counties. Lans C, Turner N, Khan T and Brauer G (2007). Waller PJ, Bernes G, Thamsborg SM, Sukura A, Ethnoveterinary medicines used to treat Richter SH, Ingebrigtsen K and Hoglund J endoparasites and stomach problems in pigs and (2001). Plants as De-Worming Agents of pets in British Columbia, Canada. Veterinary Livestock in the Nordic Countries: Historical Parasitology, 148: 325-340. Perspective, Popular Beliefs and Prospects for Laudato, M. & Capass, R. (2013). Useful plants for the Future. animal therapy. OA Alternative Medicine. 01; 1(1):1. Manthri S, Srayanthi CKS and Sidagonde S (2011). Anthelmintc Activity of Castor oil and mustard oil. Vignan Institute of Pharmaceutical, Vignan Hills, Deshmuki Village, pochapally Mandal Nalgonda District, Andhra Pradesh-508284. Mcclure SJ (2000). Sheep immunity to gastrointestinal nematode parasites: A review. Available at: http://www.csiro.au/scips. (Accessed on December 12, 2003). NFYP (2007-2012). Mannual for Mewang Geog. NCAH (2013). Annual report for National Care for Animal Health. Serbithang, Thimphu.
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Full length paper bursal disease outbreak in vaccinated young poultry flocks in southern districts of Bhutan
RATNA B GURUNG1*, SONAM TENZIN1, RINZIN PEM2, NAR K THAPA1, TENZIN GEMBO3 AND KINZANG DUKPA1
1National Centre for Animal Health, Department of Livestock, Ministry of Agriculture and Forests, Serbithang, Bhutan 2Regional Livestock Development Centre, Department of Livestock, Ministry of Agriculture and Forests, Wangdue, Bhutan 3Satellite Veterinary Laboratory, Department of Livestock, Ministry of Agriculture and Forests, Gelephu, Bhutan
*Author for correspondence: [email protected]
Copyright © 2018 Ratna B Gurung. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: Infectious Bursal Disease (IBD), also known as Gumboro disease caused by avibirnavirus belonging to the family Birnaviridae, is an economically significant poultry disease. Vaccination is considered as an important control measure depending on the strains of virus used in the vaccine. In Bhutan, with the intensification of poultry rearing activities in the country, IBD is considered economically important disease to prevent loss to poultry rearing farmers. In fiscal year 2014-15, southern districts of Bhutan bordering with India experienced series of IBD outbreaks. In an outbreak that lasted for about four months, a total of 13032 birds died in 47 farms in five districts with an average district mortality of 28.4±6.97 (2.5–44.0). An investigation on the outbreak was undertaken with the objectives to ascertain the possible factors attributing to the outbreak and provide technical recommendations to prevent outbreaks in future. The investigation found that the vaccine used before the outbreak failed to provide immune protection and confirmed the involvement of circulating virulent strain of IBD virus in the affected farms. Subsequently, the disease was controlled after vaccine was replaced.
Keywords: Infectious Bursal Disease; outbreak; investigation; vaccine; control.
1. INTRODUCTION of temperatures, disinfectants and pH (Van den Berg and Meulemans 1991; Winterfield et al. Infectious Bursal Disease (IBD) is a highly 1972). It is therefore very stable to environmental contagious acute viral disease that affects young exposure and can survive up to four months. chickens. The disease was first described by Disinfectant preparations belonging to Cosgrove (1962) in Gumboro county, Delaware formaldehyde, glutaraldehyde, chlorine and USA. Thus, this disease is also known as Gumboro iodophore groups can effectively destroy the virus. disease. IBD is a serious concern in the poultry The predilection site of IBD virus is the Bursa of industry and is considered economically significant Fabricius (BF) where the immature B lymphocytes poultry disease (Shane et al. 1994). It is caused by a are the target cells for virus replication. Immature B small, hardy avibirnavirus belonging to the family lymphocytes in BF differentiate into active immune Birnaviridae. The virus is resistant to a great range cells responsible for immune protection. BF is the 92
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 92-99, 2018 immune defense power house responsible for country, including ascertaining the possible risk protection of young birds where matured B factors attributing to the outbreak in order to lymphocytes are programmed to produce specific provide technical recommendations and prevent antibodies in response to infection or immunization. outbreaks in the future. IBD usually affects young chickens when BF is in developmental stages with sufficiently large 2. MATERIAL AND METHODS number of immature B lymphocytes. When BF is The investigation process involved visiting affected damaged, immature B lymphocytes are also farms, interviewing farm owners, examination of destroyed, leading to severe histopathological affected flock for clinical manifestation, autopsy of changes in BF and other associated organs. dead and sacrificed sick birds, sample collection, Affected birds become inactive, ruffled feathered, performing serological tests and real time droopy and excrete whitish diarrhoea. BF polymerase chain reaction tests. Samples were undergoes different stages of pathological changes tested for differential diagnosis. depending upon the stages of infection. The lesions are predominantly haemorrhages and edema of BF 2.1 Interview, post-mortem, histopathology and and accumulation of transudate. Urate diathesis is sample collection also a commonly associated renal pathological During the visit, farmers and government farm change in IBD affected birds. A sudden mortality is officials were asked about the clinical observed after brief illness in a morbid flock, which manifestation, onset of outbreak and mortality, may be as high as 20-30% (Ley et al. 1983). breed and age affected birds, feeding and treatment However, mortality in flocks challenged with regime, vaccination record and farm bio-security virulent variant IBD virus (vvIBDV) is reported as measures. Dead and sacrificed sick birds were high as 80% (Segal 2004). Vaccination is examined for gross pathology. Lesions in affected considered as an important control measure organs were recorded. Tissue samples from affected depending on the strains of virus used in vaccine organs were examined for histopathological (Van den Berg and Meulemans 1991). Maternal changes. Swab samples from cloaca and trachea derived antibody (MDA) in young chicks plays were collected while performing autopsy as well as important role of passive immunity and protection from sick birds for molecular, immunological and from infection. High maternal antibody titre in virological analysis. Bursa of Fabricius was also young chicks interferes with vaccine, leading to collected for histopathological examination. Serum immunosuppression and predisposition to infection samples were collected from infected, uninfected (Sharma et al. 1989; Naqi et al. 1983). In spite of and parent flocks for serological investigation. practising different vaccination schedules, there are reports of frequent outbreaks of IBD (Moraes et al. 2.2 Field rapid and laboratory tests 2005). Antigen detection lateral flow immunoassay using The level of MDA in young chicken plays an commercial kit (Quicking Biotech, China) important role in determining the use of vaccine following manufacturer’s instruction was strain and vaccination time. Therefore, vaccination performed to determine the presence of viruses that is usually not done until the MDA titre drops to cause avian influenza type, Newcastle disease and breakthrough level depending on the strain of IBD. vaccine used. For IBD vaccination, breakthrough Sera samples were tested to detect level of MDA titre levels of ~500 and ~200 are reported as antibody in vaccinated flocks (IBD affected and vaccine breakthrough levels for intermediate plus unaffected flocks) using enzyme-linked and intermediate strains of commercial vaccine, immunosorbent assay (ELISA) (IDEXX, respectively. Montpelier, France) following manufacturer’s In Bhutan, during mid-1990s, there were instruction. According to the test protocol, samples reports of several IBD outbreaks, particularly in that revealed sample-to-positive (SP) ratio > 0.20 government poultry farms. Regular vaccination was were considered positive due to the exposure to done as a measure to control the outbreak. In the field strain IBD virus or immunisation through recent years, with the intensification of poultry vaccination. rearing activities in the country, IBD control RT-PCR test was performed using bursal swab program was prioritised to prevent loss from samples to detect the involvement of IBD virus. disease outbreak. In 2014, a series of IBD outbreaks RT-PCR test for IBD was performed at National occurred in southern districts of Bhutan bordering Institute for Animal Health, Bangkok, Thailand and India. Loss from such outbreak entailed serious National Institute of National Institute for High economic impact to poultry industry. Therefore, the Security Animal Disease, Bhopal, India. Primers aim of this study was to undertake epidemiological from the regions of gene segments A and B were investigation on the outbreak of IBD reported in the Gurung et al. (2018) 93
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 92-99, 2018 used for detection of IBD virus. RT-PCR tests for Subsequently, same strain of IBD vaccine from Flu A, subtype H5 and N1 and ND were performed different manufacturer was procured and put into at the National Centre for Animal Health, use in all poultry establishment in the affected Serbithang, Bhutan, for differential diagnosis. areas. After the introduction of new vaccine, two Primers and probes for AI and ND were supplied by groups of serum samples were collected at same Australian Animal Health Laboratory (AAHL), interval, 57 days post vaccination: a) group that Geelong, Australia. received previous vaccine and b) group that IBD vaccine that was used in the infected flock received new vaccine. Two groups of serum 3 (IBD intermediate strain, live, NLT 10 EID50) and samples were for immune protection using ELISA subsequently procured IBD vaccine containing (IDEXX, Montpelier, France). same strain, manufactured by different company 2 (IBD Intermediate strain, live, NLT ≥ 10 EID50) 3. RESULTS were compared for the presence of immunoreactive 3.1 Clinical signs and post-mortem viral protein (VP2) using rapid test kit (Quicking The sick birds appeared exhausted, prostrated, Biotech, China). Briefly, for each vaccine, twenty ruffled feathered, huddled in large number, suffered vaccine vials were randomly picked from storage with watery white diarrhoea and soiled vent region. facility to be used for in vitro immunoreactivity A total of 34 dead and sacrificed sick birds test. Samples were prepared by reconstituting from different affected farms were examined for lyophilised vaccine in manufacturer recommended postmortem lesions (Figure 1). Bursa of Fabricius and supplied diluent. Initially, manufacturer was swollen, oedematous and haemorrhagic with recommended reconstitution using supplied diluent gelatinous yellowish transudate, covering the was prepared. Subsequently, series of dilutions at serosal surface. The bursa was filled with 1:10, 1:100, 1:200 and 1:400 were prepared. Three transudate and in one case the transudate was drops of each reconstituted or diluted vaccines were solidified taking the shape of bursa. Affected BF loaded onto the sample well of the rapid test card. showed necrotic foci and petecheal haemorrhages The samples were allowed to react for 10 minutes on the mucosal surface. Haemorrhages were seen in and the result was read. Development of colour thigh and pectoral muscles. The kidneys appeared band at the designated test position “T” on test card swollen and undergone urate diathesis. was considered as positive reaction. On the other Subcutaneous petecheal haemorrhages were also hand, absence of development of colour band at observed especially on the skin of back and lumbar position “T” was considered as negative reaction. region. Few cases of haemorrhages were found on The test was considered valid if the colour band the mucosal wall at the junction of proventriculus was developed at designated control positon “C” on and gizzard. test card.
3.2 Histopathology, rapid test and RT-PCR 2.3 Comparative immune response between two The haematoxylin and eosin (H and E) stained different vaccines tissue section from the affected bursa revealed IBD vaccine, that was used prior to outbreak was histopathological changes o f edema, immediately withdrawn after confirmation of haemorrhages, loss of follicles and lymphoid cell involvement of IBD virus in the outbreak. depletion (Figure 2).
Figure 1: Gross pathological lesions in affected organs. Haemorrhagic lesions in pectoral (A) and thigh (B) muscles; oedematous BF (C); haemorrhagic BF (D) and (E); urate diathesis of kidney (F).
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Bursal and cloacal swab samples revealed Thimphu district to as high as 44% in Tsirang positive reaction in IBD rapid test, indicating the district (Table 2). involvement of IBD virus (Table 1). The test reaction from bursal swab was stronger when 3.4 Disease evolution and mortality pattern in compared with that of cloacal swab. Rapid field test beginning of outbreak and RT-PCR test did not detect the involvement of In all affected farms, no mortality was reported avian influenza and ND virus in swab samples. after the 7th day of the onset of initial mortality. In general, mortality pattern followed a typical bell- 3.3 Spatial distribution of IBD and shaped curve, in that the mortality peaked at half epidemiological features way through the curve (3rd to 4th day) with The first case of IBD was reported in the young maximum mortality and rapidly dropping to zero layer stock of National Poultry Development Centre after a week (Figure 4). (NPDC) in Sarpang district, southern Bhutan. Three The mortality appeared to persist longer in private farms located at close proximity to NPDC bigger flock, compared with the smaller flock. All were also found to be affected by outbreak with the affected birds were approximately six weeks huge mortality. In the next four months, the disease old. The affected birds were Bovans Brown layer spread to another four districts north of Sarpang in and Ross 344 broiler. the sequence of Tsirang, Wangdue, Punakha and Thimphu (Figure 3). The disease affected poultry 3.5 Diagnosis farms in five districts with varying degrees of Findings from the clinical signs and age of affected economic losses. birds, disease evolution and mortality pattern, post- A total of 13032 birds, originating from 47 mortem lesions and test results from laboratory farms in the five districts, died of the disease analysis of samples and differential diagnosis causing an overall mortality percentage of confirmed the involvement of wild strain of virulent 28.4±6.97 (2.5–44) within four months. The district IBD virus in young flock mortality at NPDC, level mortality rates ranged from as low as 2.5% in Sarpang and private farms in Bhutan.
LFIC: Lateral Flow Immunochromatography; RT-PCR: Real time PCR
Figure 2: H and E stained tissue sections of affected bursa. A: 100X resolution tissue section of bursa showing the distended interfollicular spaces and mild degree of lymphocytic depletion; B: 400X resolution tissue section with edema and congestion.
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Figure 3: Map of Bhutan depicting the districts affected with IBD (the shaded districts are the IBD- affected districts; the circles depict the location of IBD outbreaks; and the direction of arrows indicates the likely direction of spread of disease).
Table 1: Laboratory test results. followed by boosters on 14th and 28th days of age. Sample n Agent LFIC RT-PCR Vaccine was administered via ocular route using type detection dropper. The vaccine strain used for IBD vaccination was intermediate strain of IBD live Tracheal 6 AI type A Negative Negative virus. Parent flocks, which was the source of swab virus hatching eggs, were also found to be vaccinated against IBD. Based on the ELISA cut point SP ratio Tracheal 6 AI Negative Negative of 0.20, proportions of antibody positive birds in swab subtype vaccinated-affected and vaccinated-unaffected H5 virus flocks were 85% and 84%, respectively. At 6 weeks Tracheal 6 AI Negative Negative of age, the mean antibody log10 titre of vaccinated- swab subtype affected flock (3.1±0.166, n=20) was not N1 virus significantly different from that of vaccinated- unaffected flock (3.2±0.107, n=25). Previously Tracheal 6 ND virus Negative Negative published studies reported that the minimum swab antibody log10 titre required for protection from challenge by virulent wild strain IBD virus is 3.4 Cloacal 6 IBD virus Positive Positive (Moraes et al. 2005). swab With reference to this titre value, 45% of Bursal 6 IBD virus Positive Positive vaccinated-affected and 35% of vaccinated- swab unaffected birds were found to be unprotected. Corresponding antibody titres in affected and 3.6 Vaccine, vaccination and immune profile of unaffected flock were 2665±451 (12–6911; 95% vaccinated flock CI) and 2544±348 (104–5650; 95% CI), The Day-Old Chicks (DOC) were supplied from respectively. Coefficient of variations of antibody Satara hatchery, a NPDC contracted private titre in affected and unaffected flock was 76% and hatchery in Sarpang district. DOCs were vaccinated 68%, respectively, indicating the heterogeneous against Marek’s disease on 0 day. After DOCs were nature of immune profile. lifted from hatchery, vaccination against ND, IBD and fowl pox were administered at respective 3.7 In vitro immunoreactivity of vaccine sample farms. It was found that primary dose of IBD was and response to new vaccine administered when the chicks were 3 days old, Gurung et al. (2018) 96
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At manufacturer recommended reconstitution, the number of young poultry birds in government and newly procured IBD vaccine was able to produce private farms in Bhutan. Differential diagnosis strong colour band at position “T”, indicating the excluded involvement of AI and ND virus that presence of adequate amount of IBD virus cause similar pathological conditions and mortality immunoreactive protein. in birds. Interestingly, all affected birds were Weak positive reactions were also noticed in vaccinated against IBD using intermediate strain of further dilution series (1:10 and 1:100). In contrast, vaccine. Vaccination schedule followed by farm none of the dilutions or reconstitution of IBD management was expected to protect these birds vaccine used prior to outbreak was able to produce from challenge by IBD virus, which in this case had colour band at position “T” on test card, indicating failed and resulted in IBD outbreak. It appeared the lack of immunoreactive IBD virus proteins. possibility of vaccine failure punctuated by bio- The mortality rates during the outbreak were security breach that may have allowed the entry of 42.7% and 12.9% in Sarpang and Tsirang districts, virulent strain IBD virus into susceptible respectively. With the use of new IBD vaccine, the population. Live vaccines are available at different mortality rate was dramatically reduced and no degree of attenuation and cause bursal atrophy and further IBD associated mortality was reported immunosuppression. Such intrinsic characteristics (Figure 5). Additionally, a comparative immune of IBD live vaccine and quite often failing to induce profile analysis indicated that the protective protection against very virulent strain of IBDV is antibody titre of birds that received new vaccine not uncommon (Muller et al. 2012). With the recent was significantly higher (p≤0.05) than those development in IBD vaccine, research viral vector received vaccine previously. vaccine that uses VP2 protein could be a better option since such vaccines are known to have 4. DISCUSSION minimal effect on immunosuppression and interference with maternally derived antibody Findings from the investigation confirmed the (Perozo et al. 2009; Bublot et al. 2007; Le Gros et unequivocal involvement of virulent strain of IBD al. 2009). Inadequate level of protective antibody virus in the outbreak that caused mortality of large
Table 2: Mortality data in the affected districts. Affected district Number of Flock size Number died Mortality %: Mean ± SE affected farm
Sarpang 25 28835 8768 30.9 ± 4.3 (4.4 - 93.3) Tsirang 18 7347 3114 44 ± 4.7 (5.3 - 92) Punakha 2 1050 380 29.1 ± 16.4 (12.6 - 45.6) Wangdue Phodrang 1 2066 734 35.5 Thimphu 1 1400 36 2.5
450 400 NPDC (n = 5000) 350 PL Mongar (n = 800) 300 SB Subba (n = 1400) 250 200 Duttaram (n = 800) Mortality Mortality (number) 150 100 50 0 1 2 3 4 5 6 7 8 Mortality period (days)
Figure 4: Disease evolution and mortality pattern. Numbers in parenthesis are the total stock before the outbreak.
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Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 92-99, 2018 titre in vaccinated birds and undetectable level of the reason for spread of disease in five districts immunogenic viral protein in vaccine used before despite continuous efforts in vaccination. With the the outbreak clearly suggested that the vaccine did introduction of new vaccine from another not serve the purpose. manufacturer and its ability to protect birds from Immunoassay detected high proportion of infection clearly indicated that previous vaccine antibody positive (SP ratio>0.20) birds in failed to protect birds from IBD. The use of new vaccinated flocks. However, it is important to note vaccine prevented further loss to the owners of that not all birds with an SP ratio>0.20 are poultry establishments. protected from wild strain virus exposure. SP ratio is more of diagnostic significance than an indicator 60.0 Sarpang of protection from infection. Only the birds with an 50.0 Tsirang antibody log10 titre value >3.4 are considered protected. 40.0 An effective immunization programme changes the bird from being passively immune by MDA into 30.0 an actively protected bird through the development of its own antibodies in response to vaccination. 20.0
The correct timing of vaccination plays critical role Mean mortality (%) 10.0 in this operation. If the vaccine is applied too early,
while the MDA titre is too high, the vaccine is 0.0
Jul Jun
neutralised and no protection is conferred (Naqi et Oct
Sep
Apr
Dec
Aug Nov May al. 1983; Van den Berg et al. 1991). When the Figure 5: Response to vaccine replacement in primary vaccine fails, following booster has to act flocks from mainly two affected districts. The as primary vaccine to elicit protective immune bold bar in April represents the first case of response within the expected time period (Goddard mortality due to IBD; and the broken bar in et al. 1994; Islam et al. 2005; Islam et al. 2008). On August represents the time of vaccine the other hand, if the vaccine is applied too late, replacement. then an opportunity is created for field virus to infect susceptible population and an outbreak is anticipated. Published study reported that the decay 5. CONCLUSION rate of MDA half-life is about 3-4 days (Segal Based on the facts and findings from this study, 2004). Other studies have reported that MDA there are some key areas needed to be considered in persists for as long as 15-30 days (Hitchner 1971; preventing future outbreak of IBD. All poultry Wyeth and Cullen 1979; Iordanides et al. 1991; establishments must adhere to strict bio-security Yehuda et al. 2000; Paul et al. 2005). Therefore, an practices while transporting farm supplies, accurate identification of the window of equipment and consumables; control on vehicle opportunity is extremely important. In this movement and visitor; proper use of personal investigation, it did not appear the vaccination time protective equipment and appropriate disinfectant. played a role in allowing the outbreak to occur. Concerned authority must put a system in place to At six weeks of age, affected and unaffected ensure all poultry property owners have access to flocks that received three vaccinations were not quality vaccine. Compromise in vaccine quality able to achieve protective antibody log titre value 10 may lead to serious economic loss, not only from of 3.4. It is critical to ensure adequate protection of IBD but also from many other poultry diseases. young birds from IBD virus infection at that age, owing to the development of functional BF. Acknowledgements However, it is to be noted that in the absence of The investigators acknowledge the cooperation of circulating wild strain of IBD virus, even the NPDC farm management and proprietors of susceptible flock would remain unaffected. For an affected private farms in the investigation. The outbreak to occur, IBD virus need to have access to National Institute for Animal Health, Bangkok, susceptible population. This was the most likely Thailand was kind and supportive in performing case in this outbreak. RT-PCR for IBD samples. The authors also thank In vitro immunoreactivity test of vaccine all technical officers of the Laboratory Services confirmed that the vaccine used prior to outbreak Unit for supporting the analysis of samples. lacked immunogenic protein. To investigate the quality of such vaccine in detail was beyond the Conflict of interest scope of this study. Additionally, immune profile of The authors declare that there is no conflict of birds vaccinated using such vaccine did not develop interest with regard to the findings of this study. adequate level of protection and that may have been Gurung et al. (2018) 98
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REFERENCES Naqi SA, Marquez B and Sabin N (1983). Maternal antibody and its effect on infectious bursal Bublot M, Pritchard N, Le Gros FX and Goutebroze disease immunization. Avian Diseases, 27(3): S (2007): Use of vectored vaccine against 623-631. infectious bursal disease of chickens in the face Paul BK, Huque AKMF, Kabir SML, Alam J, and of high-titred maternally derived antibody. Badhy SC (2005). Evaluation of vaccination Journal of Complete Pathway, 137: S81-S84. programmes against Gumboro disease with Cosgrove AS (1962). An apparently new disease of persistence of maternally derived antibody in chickens: avian nephrosis. Avian Diseases, 6: broiler chickens. Bangladesh Journal of 385-389. Veterinary Medicine, 3(1): 13-16. Goddard RD, Wyeth PJ and Vamey WC (1994). Perozo F, Villegas AP, Fernandez R, Cruz J, and Vaccination of commercial chicks against Pritchard N (2009). Efficacy of single dose infectious bursal disease with maternally recombinant herpesvirus of turkey infectious derived antibodies. Veterinary Records, 135: bursal disease virus (IBDV) vaccination against 273-274. a variant IBDV strain. Avian Diseases, 53: 624- Hitchner SB (1971). Persistence of present IBD 628. antibody and its effect on susceptibility of Segal Y (2004). Infectious Bursal Disease young chickens. Avian Diseases, 4: 896-900. (Gumboro). Poultry Health and Management. Iordanides P, Koumpate M and Artopois P (1991). University of Melbourne, Victoria, Australia Role of maternal antibodies in preventing IBD Shane S, Lasher H and Paxton KW (1994). in chicks in the first week of life. Delteonten Economic impact of infectious bursal disease. Kitenaiatrikes Elareias, 42: 245-249. In: Proceedings of the 2nd International Islam MN, Rashid SMH, Hoque MF, Juli MSB and Symposium of Infectious Bursal Disease and Khatun M (2008). Pathogenicity of IBDV Chicken Anaemia. Rauischholzhausen, related to outbreaks in the vaccinated flocks and Germany. the causes of vaccination failure. Journal of Sharma JM, Dohms JE and Metz AL (1989). Innovation and Development Strategies, 2(3): Comparative pathogenesis of serotype 1 and 22-30. variant serotype 1 isolates of infectious bursal Islam MT, Samad MA and Hossain MI (2005). disease virus and their effect on humoral and Immunogenic response with efficacy of certain cellular immune competence of specific- Gumboro vaccines in broiler chickens. Bang. pathogen-free chickens. Avian Diseases, 33: Journal of Veterinary Medicine, 3(1): 07-12. 112-124 Le Gros FX, Dancer A, Giacomini C, Pizzoni L, Van den Berg TP and Meulemans G (1991). Acute Bublot M, Graziani M, and F Prandini, (2009). infectious bursal disease in poultry: protection Field efficacy trial of a novel HVT-IBD vector afforded by maternally derived antibodies and vaccine for 1-day-old broilers. Vaccine, 22: interference with live vaccination. Avian 592-596. Pathology, 20: 409-421. Ley DH, Yamamoto R and Bickford AA (1983). Winterfield RW, Fadly AM and Bickford A (1972): The pathogenesis of infectious bursal disease: Infectivity and distribution of infectious bursal serologic, histopathologic, and clinical disease virus in the chicken: persistence of the chemical observations. Avian Diseases, 27: virus and lesions. Avian Diseases, 16(3): 622- 1060-1085. 632. Moraes HLS, Salle CTP, Nascimento VP, Salle FO, Wyteh PJ and Cullen GA (1979). Use of an Rocha ACGP, Souza GF, Furian TQ, and inactivated IBD oil emulsion vaccine in Artencio JO (2005). Infectious Bursal Disease: commercial broiler parent chickens. Veterinary Evaluation of maternal immunity and Records, 104: 188-193. protection by vaccination of one-day old chicks Yehuda H, Goldway M, Gutter B, Micheal A, against a challenge with a very virulent virus Godfried Y, Shaaltiel Y, Levi BZ and Pitcovski isolate. Brazilian Journal of Poultry Science, J (2000). Transfer of antibodies elicited by 7(1): 51-57. baculovirus derived vp2 of a very virulent Muller H, Mundt E, Eterradossi N and Islam MR bursal disease virus strain to progeny of (2012). Current status of vaccines against commercial breeder chickens. Avian Pathology, infectious bursal disease. Avian Pathology, 291: 13-19. 42(2): 133-139.
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Full length paper Leptospirosis associated abortion in cows at National Jersey Breeding Centre, Samtse
RATNA B GURUNG1*, TENZINLA1, DENDUP TSHERING1, SANGAY TENZIN1, LUNGTEN2, DAL B CHHETRI3, SONAM WANGCHUK4, KINZANG DUKPA1
1National Centre for Animal Health, Department of Livestock, Ministry of Agriculture and Forests, Serbithang, Bhutan 2Dzongkhag Veterinary Laboratory, Department of Livestock, Ministry of Agriculture and Forests, Samtse, Bhutan 3National Jersey Breeding Centre, Department of Livestock, Ministry of Agriculture and Forests, Samtse, Bhutan 4Public Health Laboratory, Ministry of Health, Bhutan
*Author for correspondence: [email protected]
Copyright © 2018 Ratna B Gurung. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium.
ABSTRACT: Leptospirosis is an acute zoonotic infection caused by spirochete bacteria belonging to genus Leptospira. Typical signs of leptospirosis in cattle are abortion, stillbirth, weak new born calf, sudden milk drop and infertility. A cow can abort as early as fourth month of gestation but abortion during third trimester is more common. We conducted a study with the objective to describe the findings of investigation on abortion cases of cows at the National Jersey Breeding Center in Samtse district. Paired sera samples were collected 14 days apart. First set of sera samples were collected soon after the abortion (n=6) and subjected to microscopic agglutination test (MAT) against the panel of Hardjo, Pomona and Lai like serovars. Semi-structured questionnaire was used to interview farm workers. The highest rate of abortion was 15.8%. Paired sera test revealed over fourfold rise in Microscopic Agglutination Test (MAT) titre against at least one serovar of Leptospira. Leptospirosis can significantly reduce farm profitability due to loss of calves and drop in milk production. Findings on the exposure of farm worker clearly suggest importance of farm biosecurity and occupational health safety. Isolation of organism from clinical sample will provide more information on infecting serovars in Bhutan.
Keywords: Abortion; cows; infection; leptospirosis; serovar.
1. INTRODUCTION Currently, there are 25 serogroups and 300 pathogenic serovars recognized. Leptospirosis was Leptospirosis is an acute infection caused by first identified as spirochete infection as early as spirochete bacteria belonging to genus Leptospira. 1917 by Naguchi in Japan. Rodents and The two most common genomic species are insectivores are reservoir hosts for a number of Leptospira biflexa and Leptospira interrogans, the Leptospira serovars and act as source for cross- latter being the pathogenic Leptospira (Adler and species infection. In India, the first case of de la Pena Moctezuma 2010). Vast majority of the Leptospirosis was reported in 1926 from Andaman studies available are from L. interrogans. 100
Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, page 100-106, 2018 and Nicobar Islands. Only in 1940s, Leptospirosis time of abortion. Serum samples were collected at was identified as an important veterinary problem. three different time points (Time A: March 26, It has also been recognized as one of the neglected 2015; Time B: May 24, 2015 around abortion time bacterial zoonoses. and Time C: June 6, 2015, 2 weeks post Time B). Typical signs of leptospirosis in cattle are Time interval between Time A and B was two abortion, stillbirth, weak calf, sudden milk drop and months. Similarly, the time interval between Time infertility. Abortion causes majority of the losses. A B and C was two weeks. Additionally, abortion cow can abort as early as fourth month of gestation products such as placenta, foetal organs, foetal but is more common during third trimester intestinal and stomach content were also collected. (Prescott et al. 1988; Langoni et al. 1999). Unlike abortion, milk drop syndrome occurs in the early 2.4 Serovar culture and antigen preparation stage of infection. A sudden drop in milk yield Special media for Leptospira was prepared. accompanied by rise in temperature and Commonly prevalent serovars of Leptospira inappetance in milking cows should be suspected of (Pomona, Hardjo and Lai like) were cultured at recent exposure. Abortion due to serovar hardjo Human Public Health Laboratory, Thimphu. (Kirkbride and Johnson 1989; Ellis et al. 1985) Culture was enumerated and antigen prepared. tends to be sporadic as opposed to abortion storm associated with Pomona or grippotyphosa serovars 2.5 Laboratory analysis (Grooms 2006; Genc et al. 2005). 2.5.1 Serology In Bhutan, recently conducted risk based sero- Serology was performed using Microscopic surveillance in cattle reported Leptospirosis sero- Agglutination Test (MAT), the World Health prevalence as high as 51% in some of the districts Organization (WHO) and World Animal Health (Tenzin, unpublished data). The report suggested Organization (OIE) reference method for detection that large proportions of Bhutanese cattle are of Leptospirosis in human and animals, exposed to Leptospirosis. Higher prevalence rate respectively. In microtitre plate (Nunc MaxiSorp, was reported from Punakha, Wangdue and Thermo Scientific, USA), serovar specific titrated Trashigang compared to other districts. antigen was reacted with serially diluted serum In the late spring of 2015, National Jersey samples to allow agglutination between antibody Breeding Centre, Samtse, a nucleus breeding farm, (serum sample) and antigen (serovar). The MAT reported an unusual abortion in Jersey and Karan was read by dark-field microscopy. The end point Fries cows. Subsequently, the National Centre for titre was recorded as the highest dilution of serum Animal Health, Serbithang investigated the at which 50% agglutination occurred due to antigen abortion cases in NJBC cows. The investigation and serum antibody reactivity. The four fold (1/40) confirmed abortion as associated with Leptospira and 10 fold (1/100) increase in end point titre in infection. This study describes the detail findings. samples collected 2 weeks apart (Time point B and C) were considered the past and recent exposure, 2. MATERIALS AND METHODS respectively. Similarly, the serum samples (paired sera) were also tested using enzyme linked 2.1 Veterinary inspection of aborted cows immunosorbent assay (ELISA) (IDEXX, The investigation team visited the farm and Montpelier, France) for antibody against Brucella inspected all the cows that aborted recently. Cows exposure. A sample-to-positive (SP) value ≥110% were examined for general health condition, was considered positive to antibody against management and gynecological status, including Brucella. A significant increase (p<0.05) in SP% uterine infection. for samples collected 2 weeks apart (Time point B
and C) was considered recent exposure. 2.2 Interview of farm staff and data collection
All relevant information such as history of abortion 2.5.2 Molecular test and details of affected animals; clinical Real time-polymerase chain reaction (RT-PCR) manifestation in affected animals; associated risk was performed for samples (placenta and vaginal factors of abortion; and public health were swab), using Brucella abortus and Leptospira collected using semi-structured questionnaire. The interrogans specific primers and probes. major part of the information collected was for the Amplification signal cut-off value of positive last eleven months (July 2014-May 2015). control was set at 23 cycle threshold (Ct) for the
presence of genomic material belonging to the 2.3 Sample pathogen of interest. Samples were collected from cows that aborted recently. Types of sample included vaginal swab, 2.6 Data analysis uterine discharge and urine collected around the Gurung et al. (2018) 101
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Statistical analyses and illustrations were and 15.8%) for both the periods were in the month performed using Prism 5 (GraphPad Prism of April. Software, Sandiego California, USA). Sero- reactivity between the time points was compared 3.2 Gestation period at abortion using Mann-Whitney test. Average age of the cows affected by abortion was 4.3 years. Proportions of animals that aborted in 3. RESULTS second and third trimester of gestation were 37.5% and 62.5%, respectively. Abortion was not recorded 3.1 Abortion trend at NJBC, Samtse for last 2 in the first trimester of gestation (Table 1). Majority years of abortions were observed in Trimester 3 or in the Abortion data for the period 2013-14 (July-June) beginning of Trimester 3 (Figure 2). Trimester was and 2014-15 (July-May) were compared (Figure 1). calculated as proportion of gestation days at For the period 2013-14, the mean percentage of abortion to gestation period (270 days) multiplied abortion was 2.2±0.8: 95% CI (0-7.7: maximum- by 3 and the values categorized into three minimum). The mean abortion percentage for the trimesters: 0–1 (Trimester 1); 1 – 2 (Trimester 2) period 2014-15, that included current abortion, was and 2 – 3 (Trimester 3). 5.7±1.7: 95% CI (0–15.8: maximum-minimum).
The rate of abortion for the period 2014-15 was 3.3 Rainfall data about 2.6 times higher than that of 2013-14. Leptospirosis is predominantly encountered in Interestingly, the highest rates of abortion (7.7% tropical areas with abundant rainfall. Previous studies (ref) reported that rainfall, along with 18.0 availability of rodent reservoir, plays an important 2013-14 abortion role in the spread of infection. Two-year rainfall 16.0 (%) data did not appear to have played a role in spurring 14.0 the current spread of infection. The rainfall data for 2014-15 was similar to that of 2013-14 when 12.0 abortion case was not reported in the farm (Figure 10.0 3).
Abortion Abortion (%) 8.0 3.4 Milk production trend 6.0 Daily milk production trends for the period July 2014 to May 2015 for both Jersey (n=74) and 4.0 Karan Fries (KF) (n=27) cows were compiled and analysed (Figure 2). For both breeds, about 50% 2.0 drop in milk was observed at the end of January 0.0 2015. The drop in milk yield continued for Jul Sep Nov Jan Mar May subsequent two months for KF but recovered from the beginning of April 2015. However, unlike in Figure 1: Abortion trend at NJBC, Samtse. KF, the drop in milk yield for Jersey cows did not recover in the subsequent months (Figure 4).
Table 1: Temporal data of abortion. Cow* Age ** Successful AI date Abortion date Gestation stages at abortion Day Month Trimester***
509KF 3.0 18.11.2014 23.04.2015 156 5.2 1.73 204J 7.3 18.10.2014 17.05.2015 211 7.0 2.34 503KF 3.1 10.11.2014 10.05.2015 181 6.0 2.01 363J 4.0 15.10.2014 06.04.2015 173 5.8 1.92 521KF 4.9 25.08.2014 07.03.2015 194 6.5 2.16 388J 3.6 14.11.2014 21.05.2015 188 6.3 2.09 324J 4.8 18.10.2014 06.04.2015 170 5.7 1.89 373J 3.6 23.10.2014 30.05.2015 221 7.4 2.46 *Karan Fries (KF), Jersey (J); **Years; Artificial insemination (AI); ***0 Gurung et al. (2018) 102 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, page 100-106, 2018 25 Jersey milk (July 2014-May 2015) (n=74) Minimum Maximum 20 Average 15 10 5 Daily milk production (litre)production milk Daily 0 1 11 21 31 41 51 61 71 81 91 201 101 111 121 131 141 151 161 171 181 191 211 221 231 241 251 261 271 281 291 301 311 321 331 Days in milk Figure 2: Timeline of abortion. Each colored cell is the date of successful artificial insemination, gestation and abortion. First date of successful AI was 25.8.2014. Gestation is divided into three trimesters and terminates at abortion. 1400 Myriad of farm activities were reported to have 2013-14 taken place in and around the time of this milk 1200 2014-15 drop. These activities were: all the animals in the farm received Foot and Mouth Disease vaccine on 1000 Jan 7, 2015; silage feeding began on Jan 16, 2015; 800 and animals were vaccinated against Haemorrhagic Septicemia on March 20, 2015. From the end of 600 January to first week of February 2015, the Rainfall (mm) Rainfall veterinary health record also showed several cases 400 of mastitis and wound treatment. 200 3.5 Serology MAT: In March, April and May 2015, there were 0 nine abortions in total. Since these abortions were Jul spread over the period of three months, serum Jan Jun Oct Sep Feb Apr Dec Mar Aug Nov May samples for all three different time points were Figure 3: Rainfall data (2013-14 and 2014-15) of available only from six animals that aborted. All NJBC, Samtse. the samples collected two weeks post abortion 20 Karanfries milk (July 2014-May 2015) (n=27) Minimum Maximum 15 10 5 Daily milk production (litre)production milk Daily 0 1 11 21 31 41 51 61 71 81 91 281 321 101 111 121 131 141 151 161 171 181 191 201 211 221 231 241 251 261 271 291 301 311 331 Days in milk Figure 4: Milk production trend at NJBC, Samtse Gurung et al. (2018) 103 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, page 100-106, 2018 (Time C) showed MAT titre of greater than 4. DISCUSSION fourfold when compared with that collected around Leptospirosis is a spirochete infection that has the time of abortion (Figure 5). Majority of the titre significant zoonotic importance. Exposure to values were greater than 100, indicating recent Leptospira is influenced primarily by the presence infection. Serovar Hardjo showed the highest of pathogen in the environment, persistent herd reactivity followed by Lai like and Pomona. Thus, mate shedder as potential source, rodent population the MAT titre of Time C was significantly higher in the premises, rainfall, and stress conditions in the than Time points B and A. host species. Abortion rate in cattle is dependent on the time of exposure during gestation. Generally, Brucellosis ELISA: The sera samples collected at clinical signs are manifested seven days after the Time B and Time C (paired sera) showed high exposure. Clinical signs in cows are characterized SP%. However, SP% of sera from Time C did not by fever, inappetance, conjunctivitis and bleeding. show significant increase from that of Time B. In milking cows, these signs are accompanied by sudden drop in milk yield, soft and flabby udder a and mastitis. In pregnant cows, abortion occurs 300 a* approximately after ten weeks, following exposure to infective dose (Grooms 2006). Majority of the 250 Pomona abortion occurs at third trimester of gestation. A 200 Hardjo detectable antibody titre is developed by eleventh 150 week post infection. In this study, the sudden drop in milk yield at 100 b the end January 2015 appeared to have been the 50 point of exposure that led to sudden increase in Mean MAT titre (n=6) titre MAT Mean 0 abortion rate at NJBC. The disease chronology Time A Time B Time C conformed to all the events that transpired through the period of exposure till abortion. The rainfall data during the year of abortion and the preceding C204 C324 C363 year did not show significant difference. Although studies have shown that rainfall plays important 250 a a b* role in the spread of Leptospira infection (Simoes et 200 al. 1969; Estavoyer et al. 1980; Chen 1985), it may not have influenced this outbreak. However, there 150 is a possibility that myriad of activities taking place 100 at the farm between January and February 2015 SP (%) SP may have put susceptible animals under 50 considerable level of stress, thus predisposing to 0 exposure. Therefore, it is important that the farm Time A Time B Time C management considers spreading out activities to avoid such incidence in future. Figure 5: MAT titre and Brucellosis ELISA. Infected animals are known to shed organism in asignificant (p<0.05); bnot significant (p>0.05); urine. This investigation tested only placenta and *paired sera. uterine discharge. Low detection of Leptospira genomic materials by molecular analysis may be attributed to inappropriate sampling. Future RT-PCR investigation should focus on testing urine sample Placenta sample from recently aborted cow was from aborted animals for detection of Leptospira. tested positive for Leptospira with detectable One interesting finding from this study was the signals at cut-off point Ct value of 23. All other pattern of milk yield over a period of eleven samples tested negative for Leptospira. Similarly, months. Sudden drop in milk yield was observed all samples were tested negative for Brucella by towards the end of January 2015 in both the breeds. RT-PCR. The drop continued for two months in Karan Fries Based on the findings from serological and breed and regained to fit into normal lactation molecular investigation, the abortion storm in cows curve. However, the drop in Jersey breed failed to of NJBC, Samtse may be associated with recover. This difference was not well understood Leptospirosis. Additionally, majority of the but one possible explanation could be in the stages abortions occurred in the third trimester of of lactation that this investigation captured. Milk gestation, which is typical of leptospirosis. data collected for eleven months revealed that Gurung et al. (2018) 104 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, page 100-106, 2018 majority of Jersey cows had attained peak taken to minimize the possibility of damage to production by July 2014 and steadily decreased peripheral nerves. over the period of eleven months’ record as • Streptomycin may impair fetal development opposed to KF breed milk data that steadily when administered to a pregnant animal increased for the same period of time. Perhaps, this because streptomycin readily crosses the steadily increasing trend of milk yield in KF breed placental barrier. Caution in use of this drug is was able to regain yield, despite a drop for two important to prevent ototoxicity in the fetus. months in February and March, 2015. • Treatment with streptomycin may not produce During the entire sequence of events from expected result especially when leptospirosis exposure to abortion, many farm personnel were has already done damage to the endometrium. involved in direct handling of infected animals. Under such circumstances, abortion will take Occupational health safety needs to be considered place inadvertently. to protect farm workers from being exposed to such • Withholding period of milk should be observed zoonotic infection. Through the questionnaire based on the use of specific antibiotic. In the survey, it was learnt that the farm workers did not case of use of streptomycin, a withholding experience any possible signs of Leptospirosis, but period of 72 hours should be observed before it it is important to screen them for any cases of sero- is declared safe for human consumption. positivity. In humans, the response to exposure to leptospirosis can be easily mistaken with the signs Use of antibiotics of common flu. • All clinically affected animals should be treated Leptospirosis in animals can be cured if with antibiotic (dihydro-streptomycin or detected in time. There is a range of antibiotics that streptomycin), following dose rate for are effective against Leptospira species. Top of the veterinary use. If the choice of antibiotic is not order of choice of antibiotics is available, the farm management in liaison with dihydrostreptomycin/streptomycin, followed by the National Centre for Animal Health, doxycycline, penicillin G and tetracycline. In many Serbithang should discuss and source for countries, antibiotic therapy complemented with immediate procurement. vaccination has proven to be effective in control of Leptospirosis in cattle (Russell 1958; McClain et al. 5.3 Public Health 1984; Guidugli et al. 2000). To some extent, • NCAH should notify DoPH, MoH and Samtse streptomycin and dihydrostreptomycin are also Hospital, regarding the current outbreak of effective against brucellosis. In absence of test that leptospirosis in the NJBC, Samtse. differentiates infected from vaccinated animals • Until the antibiotic therapy is initiated, the farm (DIVA) for Brucellosis, this investigation could not management should notify all the consumers to conclude whether the observed sero-reactivity was boil the farm supplied milk properly before from infection or vaccination. consumption. Unpublished data from previous study on sero- • Leptospirosis being one of the important prevalence in Bhutan revealed past exposure of zoonoses, all staffs that are regularly in contact cattle to numerous serovars of Leptospira species. with affected animals are advised to contact This is the first study undertaken to confirm active Samtse Hospital to screen for exposure to infection and its impact on production. However, leptospirosis. further investigation is required to determine the extent of infection and to examine if agents other 5.4 Sampling than Leptospira are involved. • All the herd mates to be sampled to understand the actual extent of infection in the population. 5. CONCLUSIONS AND RECOMMENDATIONS Serum samples collected at two time points two For the immediate control of abortion cases at weeks apart and tested by MAT to examine the NJBC, Samtse, following precautions must be rising antibody titre. noted and recommendations implemented: • Cattle in the areas of close proximity to the farm needs to be sampled to establish the Precautions endemicity of leptospirosis. • Care should be taken by individuals handling • The samples (serum/urine) from all the animals streptomycin for injection to avoid skin should be collected at regular interval, based on sensitivity reactions. As with all intramuscular standard protocol to assess the control program. preparations, streptomycin sulfate injection • Rodents within the farm premises should be should be injected well within the body of a trapped and samples collected to ascertain the relatively large muscle and care should be reservoir status. Gurung et al. (2018) 105 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, page 100-106, 2018 5.5 Environment management Guidugli F, Castro AA and Atallah AN (2000). • The farm should place top priority to control Antibiotics for treating leptospirosis (Cochrane the rodents in the farm structures such as feed Review), Cochrane Library, Issue 2. Update stores, silage pit and shed. Software, Oxford, U.K. • The farm management should ensure to clean Kirkbride C and Johnson M (1989). Serologic the barn/manger before and after feeding by examination of aborted ovine and bovine fetal applying power pressure water hose. This is to fluids for the diagnosis of border disease, clean rodent urine deposits in the feeding bluetongue, bovine viral diarrhea, and manger/shed and remove leftover feed to leptospiral infections. Journal of Veterinary discourage rodents visiting such areas. Diagnosis and Investigation, 1: 132-138. Langoni H, de Souza LC, da Silva AV; Luvizotto REFERENCES MC; Paes AC and Lucheis SB (1999). Incidence of leptospiral abortion in Brazilian Adler B and de la Pena Moctezuma A (2010). dairy cattle. Preventive Veterinary Medicine, Leptospira and leptospirosis. Veterinary 40: 271-275. Microbiology, 140: 287-296. McClain JBL, Ballou WR, Harrison SM and Chen T (1985). Development and present status of Steinweg DL (1984). Doxycycline therapy for leptospiral vaccine and technology of vaccine leptospirosis. Annual International Medicine, production in China. Japanese Journal of 100: 696–698. Bacteriology, 40: 755–762. Prescott J, Miller R, Nicholson V, Martin S and Ellis WA, O’Brien JJ, Bryson DG and Mackie DP Lesnick T (1988). Seroprevalence and (1985). Bovine leptospirosis: some clinical association with abortion of leptospirosis in futures of serovar hardjo infection. Veterinay cattle in Ontario. Canadian Journal of Records, 117: 101-104 Veterinary Research, 52: 210-215. Estavoyer JM, Marquelet D, Baufle GH, Becque O, Russell RWR (1958). Treatment of leptospirosis Michel-Briand Y and Pageaut G (1980). with oxytetracycline. Lancet, 2:1143–1145. Leptospirose grave avec localisationcardiaque. Simoes J, Azevedo JFD and Palmeiro JM (1969). Presse Medicine, 9: 2579. Some aspects of the Weil’s disease Genc O, Otlu S and Sahin M (2005). epidemiology based on a recent epidemic after Seroprevalence of brucellosis and leptospirosis a flood in Lisbon (1967). An. Esc. Nac. in aborted dairy cows. Turkish Journal of SaudePublica Medical Tropics, 3: 19–32. Veterinary and Animal Science, 29: 359-366. Grooms DL (2006). Reproductive losses caused by bovine viral diarrhea virus and leptospirosis. Theriogenology, 66: 624-628. Gurung et al. (2018) 106 Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 107-111, 2018 Full length paper Dairy farming enhances household income in peri- urban and rural areas of choekor in Bumthang, Bhutan GYEM TSHERING Livestock Sector, Department of Livestock, Ministry of Agriculture and Forests, Punakha, Bhutan Author for correspondence: [email protected] Copyright © 2018 Gyem Tshering. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium. ABSTRACT: A study was carried out with the objective to determine the contribution of dairy farming to the total household income in Bumthang. Choekor subdistrict was selected for the study. The study considered Lower Choekor as peri-urban areas and Upper Choekor as rural areas. A multistage sampling method was employed to select representative households from peri-urban and rural areas. A total of 100 households were randomly selected from two study areas, using the systematic random sampling method. A semi-structured survey questionnaire was used to collect both quantitative and qualitative data. The mean gross income from dairy farming in peri-urban (Nu. 63,836) was significantly higher than the rural areas (Nu. 31,918). Dairy farming was ranked as the most important source of income in peri-urban areas and second most important income source for the farmers in rural areas. Dairy contributed over 42% to the total households’ income in peri-urban areas and over 26% in rural areas. Dairy production can be further enhanced through supply of subsidized commercial feed, improvement in nutritional quality of available crop residues and development of market networks. Keywords: Dairy farming; income; peri-urban; rural. INTRODUCTION Dairy farming is an integral part of Bhutanese dairy farming was considered as the most important farming system. It is a major component of source of livelihood for the people of Bumthang Renewable Natural Resources (RNR) sector, which district. However, the documentation on contribution contributes to rural economy and poverty alleviation. of dairy farming to the total household income, The contribution of dairy sector to the Gross particularly in Bumthang district, is limited, and Domestic Product (GDP), exclusive of manure and those available are not useful (Phanchung et al. draft power, was reported to be 8% exclusive of the 2002). Further, a need for systematic research and important livestock functions (Roder 2001). documentation on small dairy farming was Traditionally, dairy measures the wealth and plays emphasized (Dorji et al. 2007; Derville and Tenzin an important role in household security and finance 2007). Therefore, the study objective was to (Luethi 1999). In Bhutan, the total dairy cattle determine the contribution of dairy farming to the population estimated was 325,628 heads (RGoB total household income in peri-urban and rural areas 2008), owned by about 77% households in the in Choekor subdistrict of Bumthang. country. Since the inception of first Five Year Plan, 107 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 107-111, 2018 1. MATERIALS AND METHODS The mean total gross income of household from different sources of income in peri-urban areas 2.1 Study areas (Nu.128,563) was not significantly different Choekor subdistrict under Bumthang district was (t =1.315, p≤0.05) from rural areas (Nu. 123,198). selected for the study. Bumthang is one of the (98) The mean gross income from dairy farming in the priority districts identified for dairy development in peri-urban area (Nu. 63,836) was significantly the country. The subdistrict is divided into Upper and higher (t =3.558, p≤0.01) than rural areas (Nu. Lower Choekor, wherein the upper Choekor (98) 31,918) (Table 1). The higher income from dairy represents rural areas and lower Choekor represents farming for the farmers in peri-urban area could be Peri-urban area, considering accessibility of milk to attributed to more milk production per household, markets or milk processing unit. which is mainly due to different milking and management practices in the study areas. It is also 2.2 Sampling method interesting to note that farmers in peri-urban owned A total of 50 farmers each was selected from upper small farms and were mostly involved in dairy and lower Choekor, using a multistage sampling farming as the main source of income generation technique. In the first stage, all villages were listed. since farmers had limited land for expansion of There were 22 villages in peri-urban and 21 villages agricultural activities. It is in agreement with Singh in rural areas. Among the enlisted villages, eight and Maharjan (2009) who reported similar case villages were randomly selected from each study between small and large farms. On the contrary, the area. In the second stage, all dairy farmers in selected lack of organized market for the sale of milk and eight villages for both study areas were enlisted. milk products in rural areas could have discouraged There were about 180 dairy farmers in peri-urban farmers to take up dairy as a primary activity, area and 174 dairy farmers in rural area. From the therefore, dairy production was more oriented enlisted dairy farmers, 50 households each were towards meeting the domestic requirements only. randomly selected from both study sites. Income from agriculture farming in peri-urban areas (Nu. 52,200) was significantly higher than the 2.3 Data analysis rural areas (Nu. 27,488). This difference could be The dataset was statistically analyzed using SPSS attributed to increasing sale of vegetables and potato software version 16. T-tests were performed in the peri-urban areas as compared with rural areas. wherever required to test significant differences in Cordyceps collection was the main income parameters between peri-urban and rural areas. The generating activity for the farmers in rural areas map for the study area was generated using (Figure 1). The mean gross income per household Geographical Information System (GIS) software – from cordyceps collection in peri-urban (Nu. 43,000) ARC MAP 9.3. was significantly lower (t =.972, p≤0.05) than the (46) rural areas (Nu. 72,050). The legalization of high- 2. RESULTS AND DISCUSSION value cordyceps gave more importance to cordyceps collection for income generation, which is apparent 3.1 Household income sources in the study areas from the contribution made by cordyceps to the total Different sources of income for peri-urban and rural household income. Tashi (2010) reported similar areas are presented in Table 1. The main income finding from western Bhutan. generating activities in the study areas were broadly There was a significant correlation (p≤0.01, classified into four main groups viz. dairy, r=0.418) between herd size and income from dairy agriculture, cordyceps, and off-farm activities. farming, indicating that large herds earned more Agricultural farming included field crops such as income as compared to small herds (Table 2). buckwheat and horticultural crops like potato, vegetables, and other farm-related activities. Off- 3.2 Contribution to household income farm activities included business, contracts, weaving Figure 1 presents the contributions of dairy farming and other jobs performed outside the farm to and other activities to the total household income in generate income. Table 1: Comparison of income between peri-urban and rural areas. Income sources t df Peri-urban Rural Significance Dairy farming 3.558 98 Nu. 63836 Nu. 31918 *** Agriculture farming 3.284 71 Nu. 52200 Nu. 27488 ** Cordyceps collection 0.972 44 Nu. 43000 Nu. 72050 ** **p≤0.01, ***p≤0.001, ns: non-significant Tshering (2018) 108 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 107-111, 2018 peri-urban and rural areas. Dairy farming contributed Gyamtsho (2000) reported income from dairy significantly to the household income in both the production up to 5% for Bumthang district. This study areas, although income from dairy farming shows that the income from dairy farming has was second to income generated from the sale of increased by around nine folds over a period of 11 cordyceps in rural areas. The contribution of dairy years, which may be due to improved breeding farming to the household income in peri-urban was systems, management practices, and animal health 42.2% and 26.2% in rural areas. These figures are, services in the study areas. A significant contribution however, exclusive of the important livestock of dairy farming to household income has also been functions in terms of draft power and manure. reported in South-east Asian countries and dairy has Current finding in the peri-urban area is comparable potential for poverty alleviation (Muriuki et al. with the findings of Phanchung et al. (2002) who 2001). reported that dairy farming contributed as high as 50% to the total household income in livestock 3.2 Farmers’ perception of income from dairy intensive areas of Chukha, Bumthang and Thimphu farming districts. The contribution from dairy farming in Table 3 provides the farmers’ perception on income rural area is also comparable with the findings of generated from dairy farming in both the study areas. Joshi (1992) and Tamang (2005) who reported that The majority of rural households (64%) felt that dairy farming contributed about 21.2% and 22% of dairy farming did not contribute significantly to the total household income in the mixed farming system, total household income; whereas majority in peri- respectively. urban households (74%) felt that dairy farming is an 50 Peri-urban 45 Rural 40 35 30 25 20 15 10 5 Contribution to household income (%) income tohousehold Contribution 0 Cordyceps Dairy Agriculture Off-farm Sources of income Figure 1: Contribution of dairy farming and other activities to total household income. Table 2: Correlation between herd size, farm size important activity that contributed to the total and income from dairy farming in the study areas. household income. The majority of peri-urban households were Variables Herd size farm size Income divided on whether the income from dairy farming Herd size 1 0.154 0.42** has remain unchanged or decreased over the years Farm size 1 0.02 (Table 4). Similar views were expressed by Income 1 respondents in rural areas that the income from dairy farming has not changed. However, the majority **p≤0.01 Table 3: Farmers’ perception on income from dairy farming. Contribution of dairy to total household income Area n no income <25% 25-50% 50-75% >75% Peri-urban 50 26% 32% 16% 10% 16% Rural area 50 64% 26% 8% 2% 0% Tshering (2018) 109 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 107-111, 2018 were of the view that the number of households dairy as a profitable business but prefer taking up rearing dairy cattle has increased over the years. government jobs, business, contract works and plying taxis. Family division and separation were Table 4: Farmers’ perceptions on trends of dairy also reported as a reason causing labor shortage in a farming and income (%). farm household (Tamang 2005). Respondents 3. CONCLUSION Variables Trend Peri- Rural The contribution of dairy farming to the total urban areas household income was significant at household level (n=50) (n=50) in both the study areas. The contribution of dairy Income Increased 6% 16% farming to household income is higher in peri-urban Decreased 46% 18% areas, compared with rural areas. Cattle holding, No change 46% 66% number of milking animals and breed composition per household do not affect the household milk No idea 2% 0% production and income. Different milking practices Households Increased 88% 72% and poor management are the main factors affecting rearing cattle Decreased 10% 06% milk production and income generation between No change 02% 22% peri-urban areas and rural areas. Therefore, there is a need to create awareness of milking practices and improved dairy husbandry practices among rural 3.4 Constraints to dairy production population. Shortage of fodder and labour are the The constraints to dairy farming, as perceived by main constraints hindering dairy production in the respondents, are presented in Table 5. Fodder study areas. To make dairy farming attractive to the shortage was mentioned as a major constraint by people in rural areas, the labor-saving devices such both peri-urban and rural farmers, which could be as cream separators, portable machines, churners etc. attributed to limited landholding for growing grasses should be encouraged. There is a need to provide for dairy cattle. This is in agreement with Phanchung commercial feed through subsidy program, improve et al. (2001) who reported that shortage of fodder is nutritional quality of available crop residues and the most important constraint hindering dairy develop market network to enhance dairy production production. Fodder production receives lower and improve livelihood of farmers. priority compared with field crops. The finding also concurs with that of Roder et al. (1998) who reported Acknowledgements that less priority is given to fodder development due We are thankful to the Department of Livestock to competition from other crops. (DoL) for the generous financial support to conduct The second most important constraint to dairy this study. We also acknowledge the support production, according to peri-urban farmers, was provided by the management of CNR and RNR- labor shortage in the household. This could be RDC, Jakar. The cooperation and support of rural attributed to increasing number of children being communities of Choekor subdistrict are highly enrolled in schools due to high priority given to acknowledged. Table 5: Constraints to dairy farming. Study areas N Feed shortage Labor shortage Financial Marketing Peri-urban 48 47.9% 47.9% 4.2% 0.0% Rural areas 41 60.4% 14.6% 0.0% 25.0% Total 89 59.1% 32.6% 2.1% 12.5% education. Moreover, after education, most family REFERENCES members get employed in government and private Dervile M and Tenzin G (2007). Dairy Chain organizations away from home and they cannot Analysis. Ministry of Agriculture, Thimphu. extend their help to the needy parents (Tamang Devendra C and Thomas D (2002). Crop-animal 2005). Generally, most youths migrate to urban areas systems in Asia: Importance of livestock and to seek better opportunities, which also contributes characterization of agro-ecological zones. to labor shortage. UNDP (2011) reported that rural- Agricultural Systems, 6: 5-15. urban migration in the country is one of the highest Dorji J, Tshering G, Sherpa DL, and Bhujel P (2007). in the South Asian region. Dorji et al. (2007) also Smallholder Dairy Farming Practices in Peri- reported that younger generations do not perceive Tshering (2018) 110 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 107-111, 2018 urban areas of Chamkhar. RNR-RC, Jakar, Singh M and Maharjan KL (2009). Dairy Production Bumthang. and Implication in the Household Income in Gyamtsho P (2000). The economy of Yak. Journal of Terai Region of Nepal: A Case study of Chitwan Bhutan Studies, 2 (1): 52-56. district. CNAS Journal, 32 (2): 213-141. Joshi BR (1992). The role of large ruminants. In: Tamang NB (2005). Comparative milk yield of Abington J, Sustainable Livestock Production in Mithun-Siri cross and Siri cattle managed on the Mountain Agro-ecosystem of Nepal. Food village farms in Bhutan. Victoria, Australia: and Agriculture Organization of UN, Rome: 47- University of Melbourne. 75. Tashi S (2010). Sustainable Livelihoods of the Leuthi N (1999). Bovine and Equine in Bhutan. People in the Alpine Region of Western Bhutan. Bumthang: RNR-RC, Jakar, Special Publication Proceedings of the seminar on sustainable rural No. 2. development. College of Natural Resources MoAF (2010). Agriculture Commercialisation and (CNR), Lobesa, Punakha: 23-30. diversification in Bhutan. SD-IFRI, Thimphu. Muriuki HG, Mwangi DM, and Thorpe W (2001). How smallholder dairy systems in Kenya contribute to food security and poverty alleviation. Proceedings of the 28th scientific conference of Tanzania Society of Animal Production, Morogoro, Tanzania: 7-9. Phanchung, Dorji P, Sonam T, and Pelden K (2002). Smallholder Dairy in Bhutan. Characteristics, constraints and Development Opportunities. In: Tulchand M, Smallholder Dairy in Mixed Farming Systems of the HKH (pp. 22-35). Hill Sede Press (P) Ltd., Kathmandu, Nepal. Tshering (2018) 111 Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 112-114, 2018 Short Communication Microbial load in local and imported raw milk TENZIN PENJOR1* AND TSHERING GYELTSHEN2 1National Piggery Research Centre, Department of Livestock, Ministry of Agriculture and Forest, Gelephu, Thimphu, Bhutan 2College of Natural Resources, Royal University of Bhutan, Lobesa, Punakha, Bhutan *Author for correspondence: [email protected] Copyright © 2018 Tenzin Penjor. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium. ABSTRACT: There was a complaint on supplying low quality milk to residents of Samdrup Jongkhar. This study was carried out in winter to evaluate and compare the milk quality from two main sources. The emphasis was given more on assessing the microbial load in raw milk for both local and imported milk. Samples from individual cans (N=35) and bulk tank (N=7) were collected for imported and local milk. A volume of 50 ml milk sample was collected aseptically in a 50 ml vial. The sample was transported to laboratory at 2- 8⁰C, packed in cool box with ice packs. The microbial load in milk was estimated using Pour Plate Technique at 30±2⁰C at 72 hours. The microbial load was calculated in cfu ml-1. All microbial counts were converted to the base 10 logarithm of the number of colony forming units per ml of milk samples (log cfu ml-1). T-test was conducted to compare the microbial loads between imported and local milk. The microbial load was significantly higher in imported milk (M=8.54±0.06) when compared with local milk (M=8.39±0.03). The mean values of microbial load for imported (N=35) and local milk -1 -1 (N=35) were 8.54 log10 cfu ml and 8.39 log10 cfu ml , respectively. The mean microbial load for both imported and local milk exceeded the EU and US standard. The mean microbial load for bulk tank samples was higher, compared with individual can samples from both sources. Timely monitoring of milk at producer’s level, milkman and sales counter are necessary to minimize contamination of milk. Keywords: Dairy; hygiene; microbial load; milk quality; raw milk. 1. INTRODUCTION 2010). Lack of hygiene, adulteration with various agents and improper cold chain facilities are primary Milk is the best and cheapest source of nutrition. It contributors to low milk quality (Zia et al. 2011). is one of the daily diets, easily accepted and used by Recently, there has been dissatisfaction among all age groups in the mankind (Ayub et al. 2007). In consumers of milk in the district of Bhutan, milk is an important food commodity for Samdrupjongkha. The residents of Samdrup household consumption. Milk is consumed as fresh Jongkhar purchase milk from two sources and they milk and in the form of butter, cheese, yogurt and complained of low milk quality and adulteration. whey. However, the safety of milk is important for Despite complaints, no study was carried out to consumers. Milk and milk products can harbor a investigate and establish if complaints are true. variety of microorganisms and can be important Therefore, this study was undertaken with the sources of food borne pathogens. Hygienic quality objective to evaluate the milk quality in terms of of milk is of great importance since bacterial growth microbial load in raw milk. in milk can be a health hazard to consumers (Millogo 112 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 112-114, 2018 2. MATERIALS AND METHOD Bhutan by several milk men with or without proper transport facilities. The milk is usually packed in The study was conducted in Samdrup Jongkhar jerry cans, which are actually difficult for district, which is located at an altitude of 200 masl maintaining hygiene and proper sanitation. The poor and temperature ranges from 14° to 36 °C. Milk hygiene and sanitation of jerry cans could be the samples from local and imported milk were sources of contamination and microbial growth. The collected. Five samples from individual cans and one high air temperature of Assam could be other sample from bulk tank were collected for seven possible causes of milk contamination and spoilage. rounds. The samples were collected once in a week. Whereas, the local milk is packed in aluminum cans, The amount of each sample was about 50 ml. transported by proper milk van. Further, the sale Samples were packed and transported at 2-8°C counter is well equipped where optimum cold chain within 24 hours for laboratory examination. is maintained until distribution. The laboratory examination of microbial load in The microbial load in our study was higher than milk was carried out following Pour Plate Technique those reported for Pakistan and India (Reddy et al. at 32°C for 72 hours, as prescribed in the standard 2012; Saxena and Rai 2013; Mohammed et al. 2009; laboratory procedures (SOPs) (National Centre for Lingathurai and Vellathurai 2011; Khaliq et al. Animal Health 2013). The microbial load was 2001). However, the microbial load was close to calculated in cfu ml-1. All microbial counts were findings reported by Pant et al. (2013) and Hossain converted to the base 10 logarithm of the number of et al. (2011). Microbial load in milk has also been colony forming units per ml of milk samples (log cfu reported by several studies in Bangladesh, Nepal and ml-1). The data were entered in Microsoft Excel. It Malaysia regions was lower (Dey and Karim 2013; was then processed and analyzed in SPSS vs.16.0. Bhattarai and Singha 2010; Yuen et al. 2012). The Two sample t-test was used to compare microbial microbial load has been reported to be lower in load content between local and imported milk. United States, European countries and African regions (Mattias Orregard 2013; Torkar and Teger 3. RESULTS AND DISCUSSION 2008; Welearegy et al. 2012; Pantoja et al. 2009). 3.1 Total plate count The high microbial load in milk is due to inadequate The microbial load was significantly higher in sanitary conditions during milking, collections and imported milk when compared with local milk transportation of milk (Devi and Sowmya 2012; (Figure 1). The mean microbial load in bulk tank Reddy et al. 2012; Minj and Behera 2012). The samples were higher when compared with samples microbial load in milk is also affected by season and collected from individual cans for both imported time (Pantoja et al. 2009; Donkor et al. 2007; Hassan milk and local milk. The microbial load mean value et al. 2009; Muhammad et al. 2009). for both imported and local milk exceeded the European Union (EU) and United States (US) 4. CONCLUSION standard (Mc Langhlin 2006). This could be because The microbial load of both imported and local milk of difference in air temperature, hygienic condition, exceeds the EU and US standard limit. Therefore, milk cans used and difference in transportation the complaints of residents of Samdrup Jongkhar are facilities. Imported milk is usually brought inside justified. To minimize contamination, certain a regulations and regular monitoring at all stages of a 8.6 production are essential. It is also important to fix the price of milk based on quality to encourage 8.5 b b hygiene milk production and minimize contamination. 8.4 The current study focused only at one place. Similar investigation needs to be carried out at 8.3 different places and at production and marketing 8.2 stages across seasons. 8.1 REFERENCES Microbialload (Log10 cfu/ml) Ayub M, Ahmed Q, Abbas M, Qazi IM and 8 Bulk can Individual can Khattak IA (2007). Composition and Adulteration Analysis of Milk Samples. Local milk Imported milk Sarhad Journal of Agriculture, 23 (4): 35-39 Figure 1: Microbial load of individual and Bhattarai B and Singha SP (2010). Quality bulk samples of local and imported milk Evaluation of Milk at Different Levels of Milk Penjor and Gyeltshen (2018) 113 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 112-114, 2018 Chain System in Makwanpur District, Nepal. Pant R, Nirwal S and Rai N (2013). Prevalence of Journal of Food Science Technology, Nepal. 6: Antibiotic Resistant Bacteria and Analysis of 80-83. Microbial Quality of Raw Milk Samples Devi NP and Sowmya D (2012). Microbial Count Collected From Different Regions of of Raw Cow's Milk in Chennai. Dehradun. Dehradun, India: Department of Dey S and Karim MH (2013). Study on Biotechnology, Graphic Era University, physiochemical and microbial quality of Dehradun. available raw, pasteurized and VHT milk Pantoja JC, Reinemann DJ and Ruegg PL (2009). during preservation. International Journal of Associations Among Milk Quality Indicators Science Inventions Today, 4: 150- 157. in Raw bulk milk. Journal of Dairy Science, Donkor ES, Aning KG, Omore A, Nurah GK, 92: 4978 - 4987. Osapo EL and Staal S (2007). Risks Factors in Reddy MB, Sasikala P and Karthik A (2012). the Hygienic quality of milk in Ghana. The Microbial Analysis and Constituents of Mixed Open Food Science Journal, 3: 6- 9. milk samples in Rural areas of Pileru Chittoor Hassan A, Amjad I and Mahmood S (2009). District: Andhar Pradesh. International Journal Microbiological and Physiochemical analyis of Pharmaceutical, Chemical and Biological of different UHT milk available in a local Sciences, 5: 692- 695. market. Asian Journal of Food and Agro- Saxena M and Rai P (2013). Microbiological And Industry, 5: 434 - 447. Chemical Analysis of Raw, Pasteurized And Hossain T J, Alam MK and Sikdar D (2011). UHT Milk During Preservation in India. Chemical And Microbiological Quality International Journal of Chem Tech Research, Assessment of Raw and Processed Liquid 5(6): 2804- 2809. Market Milks of Bangladesh. Continental J. Torkar KG and Teger SG (2008). The Food Science and Technology, 5 (2): 6-17. Microbiological Quality of Raw Milk After Khaliq K, Ashfaque M, Hussan I and Akhtar M Introducing The Two Day's Milk Collection (2001). Bacteriological Studies on Raw Milk System. Slovenia: Department of Animal supplied to Faisalabad city During Summer Science. 61- 74 Months. Pakistan Veterinary Journal, 2: 13-17. Welearegay H, Yilma Z and Giorgis YT (2012). Lingathurai S and Vellathurai P (2011). Hygienic practices and microbiological quality Bacteriological quality and safety of raw cow of raw milk produced under different farm size milk in Mudurai, South India. in Hawassa, Southern Ethiopia. Research Mc Langhlin F (2006). A Brief Comparison of Paper. United States and European Union Standards Yuen SK, Yee CF and Yin FH ( 2012). for Fluid Dairy Production. Michigan: Microbiological Quality and the Impact of Michigan State University. Hygienic Practices on the Raw Milk Obtained Millogo V (2010). Milk Production of Hand- from the Small- scale Dairy Farmers in Sabah, Milked Dairy Cattle in Burkina Faso. Uppsala: Malaysia. International Journal of Agriculture Swedish University of Agricultural Sciences. and Food Science, 2(2): 55-59. Minj AK and Behera N (2012). A comparative Zia U, Muhamood T and Ali MR (2011). Dairy microbiological quality assessment of rural Development in Pakistan. Rome: FAO. and urban milk samples. Odisha, India: School of Life Sciences, Sambalpur University. Muhammad K, Altaf I, Hanif A, Anjun AA and Tipu MY (2009). Monitoring of Hygienic Status of raw milk marketed in Lahore city, Paskistan. The Journal of Animal and Plant Sciences 3: 67-75. National Centre for Animal Health (2013). Standard Laboratory Manual: For Veterinary Laboratories in Bhutan. Thimphu: National Centre for Animal Health. Orregard M (2013). Quality analysis of raw milk along the value chain of the informal milk market in Kiambu County, Kenya. Kiambu County: Swedish University of Agricultural Sciences. Penjor and Gyeltshen (2018) 114 Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 115-116, 2018 Case Report Surgical removal of teat siphon from cow’s udder TENZIN WANGCHUK1* AND PREM B GIRI2 1Dzongkhag Veterinary Hospital, Department of Livestock, Ministry of Agriculture and Forest, Gelephu, Thimphu, Paro, Bhutan 2Livestock Extension Centre, Department of Livestock, Ministry of Agriculture and Forest, Gelephu, Thimphu, Shari, Paro, Bhutan *Author for correspondence: [email protected] Copyright © 2018 Tenzin Wangchuk. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium. 1. CASE HISTORY A five-year-old Jersey cross breed cow at the second stage of lactation was suffering from a chronic suppurative mastitis at Damchena village under Shari subdistrict, Paro. The owner had treated the case by applying hot fomentation and other local methods but failed to cure the case. The case was then reported to the subdistrict livestock extension office for treatment on 23 November 2017. The livestock staff had examined the case and found thick purulent discharges oozing out from the teat canal while squeezing the teat and udder. Since the case was away from the subdistrict livestock extension centre, the extension staff had instructed the owner to drain out pus from the udder with the help of a teat siphon. Mammary infusions were provided. The owner had inserted the teat siphon into the teat canal on 25 November 2017, to drain out pus, as per the instruction but unfortunately the teat siphon had slipped into the mammary gland when cow moved during operation. The owner reported about the incident to the subdistrict extension staff on 27 November 2017. The case was then referred to the District Veterinary Hospital (DVH), Paro on 28 November 2017 when both the owner and the sub district livestock staff failed to remove the teat siphon manually. The Veterinary Officer from DVH, Paro visited the case and tried to remove the teat siphon manually but was in vain. We decided to remove teat siphon by performing surgery. The owner was informed about the decision of surgical intervention on the next day. The owner consented to the decision. 2. ASCERTAINING POSITION OF THE TEAT SIPHON attempt to remove the teat siphon manually after X- ray examination was unsuccessful. We requested Miss Jamie Vaughan, Maya Foundation, Paro to lend her digital X-ray machine (Ultralight 9020HF, Collimeter, EcoRay Co., LTD. SURGICAL INTERVENTIONS Seol, Korea 153-786MarkIIG® sound company) to The animal was restrained in standing position ascertain the position of the teat siphon inside the within the crush. The surgical site was prepared by udder. She kindly lent her machine and also shaving the hairs around the teat with blade and then supported us in performing the X-ray. We built cleaned the site with 5% chlorhexidine, followed by temporary crush with wooden poles and planks to povidone iodine and rectified spirit. The incision site restrain the animal during X-ray and surgery. We was desensitized by ring nerve block using 20 ml of took X-ray laterally as well as posterio-anteriorly 2% lignocaine at the base of teat. and the X-ray picture showed a very clear position About 2 cm longitudinal incision was made on of teat siphon inside the udder (Figure 1). Our the teat at the site where teat siphon was located 115 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 115-116, 2018 (based on X-ray examination). When incision Acknowledgements reached the teat canal, a thick purulent discharge We would like to thank Miss Jamie Vaughan, Maya gushed out and the remaining pus were removed by Foundation, Paro for lending her X-ray machine and squeezing the mammary gland. A teat siphon was also for assisting us in performing X-ray at the site. located and was removed using 14 inches curved Dr. Tenzin, Head DPCU, NCAH is acknowledged artery forceps. The quarter was flushed with for helping us to draft and edit this case report. povidone iodine, followed by metronidazole solution. The inner and external muscular layer were sutured with absorbable polyglacton (vicry l 2-0) and vicryl 1, respectively. The wound was dressed with gamma benzene hexachloride ointment. As part of post-operative treatment, the animal was provided with injection 10ml meloxicam i/v, injection ampicillin cloxacillin 2g i/v and injection 20ml oxytetracycline (LA) i/m. The animal was followed up after the operation and the surgical wound had healed. The animal also recovered from mammary infection. 3. CONCLUSION AND RECOMMENDATIONS This incident demonstrates that teat siphon should be used only by trained livestock personnel and should not be given to the farmer for intervention without technical guidance. The portable digital X-ray machine had helped to locate the teat siphon inside the mammary gland in a cow and can also be useful to diagnose certain conditions like fracture and foreign objects in animals. Therefore, X-ray machine is a critical equipment for the district veterinary hospitals. Wangchuk and Giri (2018) 116 Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 117-119, 2018 Short Communication Effects of body condition score on postpartum oestrus cycle in dairy cows PEMA UGYEN1*, PENJOR2 AND RINZIN DORJI3 1District Livestock Sector, Department of Livestock, Ministry of Agriculture and Forest, Punakha, Bhutan 2College of Natural Resources, Royal University of Bhutan, Lobesa, Punakha, Bhutan 3District Veterinary Hospital, Department of Livestock, Ministry of Agriculture and Forest, Tsirang, Bhutan *Author for correspondence: [email protected]. Copyright © 2018 Pema Ugyen. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium. ABSTRACT: The study objective was to determine the effect of body condition score on postpartum oestrus cycle in dairy cows. The study was carried out in Tsirang district from November to December 2013. A total of 80 Jersey cross cows; 40 cyclic and 40 postpartum anoestrus were chosen for the study. Cows, which exhibited normal heat signs with no history of repeat breeding and nymphomania, were considered as cyclic cows. Cows, which did not show oestrus signs within 90 days after calving, were considered as postpartum anoestrus cows. The visual scoring method, using the American standard (scale one to five), was employed to judge the body condition of cows. There was a significant association between the body condition and manifestation of oestrus cycle of cows. The mean value of first postpartum oestrus was recorded at 7.13 months, which was higher than the expected first postpartum oestrus that occurs within 2-3 months after calving, to achieve optimal calving interval. The results suggest that dairy cows need minimum body condition score of 3 to 3.5, for expression of normal oestrus cycle and to produce one calf per year. Keywords: Postpartum anoestrus; Body Condition Score; oestrus; Jersey cross cows. 1. INTRODUCTION in cattle following parturition. The main factor for delayed ovulation and oestrus after calving are due Oestrus is the period when the female is receptive to limited energy intake, lower body reserves, to male in the act of mating. The range of oestrus increased partitioning of energy to milk production cycle in a cow is between 18 to 24 days with 21 and heavy suckling by calves. This nutritional days as an average (Banerjee 2010). A cow will status can be assessed by observing the body come to first heat within 30 to 45 days after calving condition score (BCS) of dairy cow. if the cow has calved normally and is under good BCS is a good technique to assess the condition management and feeding system. Cows that do not of dairy cows at regular intervals. The main come to heat within 60 to 90 days after calving can purpose of BCS is to achieve a balance between be considered as problematic or postpartum economic feeding, good production and animal anoestrus cows. Postpartum anoestrus is an welfare. This can be done at important stages of interruption in the normal cyclical production of production such as drying off, calving time, early ova from ovaries, which occurs at varying degrees lactation and at service. The amount of body fat in 117 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 117-119, 2018 animal at different stage of production cycle is an 3.1 Household size and Cattle holding important factor, which enables reproductive The mean household size was over five persons per efficiency. The display of oestrus behaviour can be house with the total of 413 people across study delayed in cows having poor BCS and those cows areas (Table 1). The result of this study also under heat stress. Different points of scale are used revealed that less than three people from every to assess BCS of dairy cows. In Bhutan, the household are working on the farm. In total, there American standard of scale from 1-5 points is were 344 heads of cattle with an average of over commonly followed; a score of 1 is assessed as four cattle heads per household. The cattle holding extremely thin or emaciated and a score of 5 as per household in the study area ranged from 1-9. being extremely obese. However, the study on effects of BCS on postpartum oestrus cycle in dairy 3.2 Milk yield and parity cows has not been undertaken in Bhutan. The mean milk yield of lactating cows in the study Therefore, this study was initiated with the area was over four kg, which is very close to the objective to evaluate the effects of BCS on oestrus mean milk yield of pure Jersey cow managed under cycle of a cow. farmers’ conditions (Phangchung et al. 2002). The mean parity of a cow was over two numbers when 2. MATERIALS AND METHOD the mean age of cow was less than six years. 2.1 Study area and data collection 3.3 Postpartum anoestrus period in cows The study was conducted in Tsirang district, The duration of postpartum anestrus condition is covering three subdistricts of Kikhorthang, being recognized as critical event in the Rangthangling and Tsholingkhar. Dairy farming reproduction of a cow. In this study, the mean value plays a vital role in generating household income, of first postpartum oestrus was recorded over seven besides poultry and agriculture farming in the months, which is much higher than the expected district. A total of 80 Jersey cross cows (40 cyclic first postpartum oestrus that occurs within two to and 40 postpartum anoestrus) were selected for the three months after calving to achieve optimal study. Convenience sampling method was used for calving interval (Kamal et al. 2014). It can be selecting both the cyclic and postpartum anoestrus explained that the higher open period directly cows because BCS from cyclic cows were done as affects the inter-calving period. Calving interval and when the cow was brought for artificial also determines the reproductive efficiency and insemination service at District Veterinary based on this result, the calving interval is much Hospital. Cows that showed normal heat signs with longer (16 to 18 months), while the suggested no history of repeat breeding and nymphomania calving interval, according to Thomas and Sastry were considered as cyclic cows. The cows that did (2000), for a cow is 12 to 13 months. The severity not show oestrus signs within 90 days after calving of negative energy balance before and after calving were considered as postpartum anoestrus cows. is the main factor delaying resumption of the first Visual scoring method was used to judge the body postpartum oestrus (Johnson et al. 2012). Thus, condition of cows. The scale of 1 was rated as during the early postpartum period, it is advisable emaciated, 2 for thin and skinny, three 3 as ideal, 4 for the dairy farmers to allow cows to maximize for obese and 5 for very obese. The household dry matter intake with fresh, palatable and high- survey was also carried out using a semi-structured quality feed having adequate energy, protein, questionnaire, consisting both closed- and open- minerals and vitamins. ended questions. The survey gathered both qualitative and quantitative information on cattle Table 1: Socio-demographic information of study holding, milk yield, parity and other management area. aspects of dairy farming at the household level. Parameters Mean SD 2.2 Data analysis Household size (nos.) 5.16 3.32 The data obtained from questionnaire survey and Farm labour available (nos.) 2.72 1.68 visual scoring method were tabulated in Microsoft Cattle holding per household (nos.) 4.30 2.13 Excel 2016 and analysed using SPSS version 16.0 Daily milk yield per cow (kg) 4.44 1.87 (Armonk, NY: IBM Corp). Chi-square test was Cow age (years) 5.69 2.63 conducted to determine the effect of cow condition Parity (nos.) 2.34 1.66 on manifestation of oestrus cycle. Descriptive Postpartum period 7.13 2.61 statistics was used to obtain means of household size, cattle holding, milk yield and parity. 3.4 Effect of cow body condition on oestrus cycle Statistically, the result showed significant 3. RESULTS AND DISCUSSION association between body condition of a cow and Ugyen et al. (2018) 118 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 117-119, 2018 manifestation of oestrus cycle (χ2 (2)=14.68, Effects of body condition and composition on p≤0.01). Table 2 presents the contingency table on the onset of postpartum ovarian activity in beef the effect of BCS on oestrus cycle of a cow. cows. Journal of Animal and Veterinary Among 40 cyclic cows; 18 cows had ideal BCS, 13 advances, 10 (24): 3236-3243. cows were fat and nine cows were thin and skinny. Johnson CR, Spencer J, Ahmadzadeh A and Dalton The result of postpartum anoestrus showed 26 cows JC (2012). How soon after calving do cows as thin and skinny, 8 cows as ideal and 6 cows as show heat and evidence of first ovulation. fat. This result indicated that optimum body Western dairy news. Moscow, Russia: condition (3–3.5) is required for expression of University of Idaho. normal oestrus cycle. Dominguez et al. (2011) Kamal MM, Bhuiyan MU, Parveen N, Momont reported similar result and claimed that BCS at HW, Shamsuddin M (2014). Risk factors for calving has association with the length of the postpartum anooestrus in crossbred cows in postpartum interval, leading to anoestrus condition. Bangladesh. Turkish Journal of Veterinary and Looper et al. (1997) reported that cows with Animal Science, 38: 151-156. moderate BCS has shorter postpartum interval to Looper ML, Lents CA, Vizcarraand JA and first normal luteal activity, compared to thin and Wettemann RP (1997). Effects of body emaciated cows. condition on luteal activity and ooestrus in postpartum beef cows. Table 2: Effect of body condition on oestrus cycle Phangchung, Dorji P, Sonam T and Pelden K of cow. (2002). Smallholder Dairy Farming in Bhutan. Category Thin and Ideal Fat Total Kathmandu:Nepal: ICIMOD. skinny (3-3.5) (>4) Thomas CK and Sastry NSR (2000). Dairy Bovine (<2.5) productin. New Delhi-110 002.ISBN 81-7096- 252-8: Kalyani Publishers. 1. Cyclic cows 9 18 13 40 2. Postpartum 26 8 6 40 Anoestrus cows Total 35 28 17 80 4. CONCLUSIONS There is strong association between body condition score and manifestation of oestrus cycle in a cow. Optimum body condition is required to express normal oestrus cycle in dairy cows. From this study, it can be speculated that body condition score is important in judging the condition of cyclic and anoestrus dairy cows. It is important to provide balanced nutritional feed and mineral supplements before and after parturition to improve reproductive performance of dairy cows and increase the number of calves born per cow. Acknowledgement The authors thank the dairy cattle owner for allowing them to use his dairy cows for the study. The authors also thank the staff of District Veterinary Hospital, Tsirang, for their assistance during field assessment and data collection. REFERENCES Banerjee GC (2010). A textbook of Animal Husbandry, 8th edn. New Delhi: Oxford & IBH Publishing Co.Pvt.Ltd. Dominguez MA, Villanueva-Castillo A, Ponce- Acosta MP, Castillo-Almaguer R, Gutierrez- Rodriguez E and Medellin-Avila O (2011). Ugyen et al. (2018) 119 Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 120-122, 2018 Short Communication Moisture content of local cottage cheese and butter in Western Bhutan SONAM YANGCHEN1*, PHUNTSHO T NORBU2 AND Y NAKANISH2 1Livestock Production Division, Department of Livestock, Ministry of Agriculture and Forest, Thimphu, Bhutan 2National Dairy Research Centre, Department of Livestock, Ministry of Agriculture and Forest, Yusipang, Thimphu, Bhutan *Corresponding Author: [email protected] Copyright © 2018 Sonam Yangchen. The original work must be properly cited to Permit unrestricted use, distribution, and reproduction of this article in any medium. ABSTRACT: A study was conducted with the objectives to determine the moisture content of cottage cheese (popularly known as datshi in Bhutan) and butter available in the market and establish baseline data to facilitate systematic grading and standardization of quality of dairy products in the country. The study was carried out in the districts of Thimphu, Paro and Haa in western region of Bhutan. Samples were randomly collected from Milk Processing Units (MPUs), dairy sales counters, retail shops, roadside vendors, and weekend vegetable markets. Samples were collected from the month of April to June, 2017. The butter samples were collected from only two districts of Thimphu and Paro. All samples were subjected to laboratory analyses. The moisture content ranged between 60.24% and 71.24% with overall average moisture content of 66.69%. The moisture content in the butter samples ranged from 13.78% to 38.79% with the average moisture content of 21.64%. The study results suggest that the moisture content of cheese is within the acceptable range but the moisture content of butter is higher than the acceptable range. Keywords: Butter; cottage cheese; dairy; datshi; milk. 1. INTRODUCTION equipment and knowledge. Production of datshi has increased from 2,300 MT in 2012 to 3,664.55 MT Cottage cheese is a high-moisture, un-ripened soft in 2016 (Livestock Statistics 2012 and 2016), cheese made from skimmed milk. This cheese is which is associated with increase in milk also made from concentrated nonfat milk or production over the years. However, it is unclear reconstituted nonfat dry milk. Cottage whether the increase in cottage cheese production cheese belongs to the class of natural, unripened, has also resulted in improvement in cheese quality. soft cheeses. Since it has a significantly lower fat This is because the manufacturing practices remain content than most cheeses and is a good source of mostly conventional or traditional. Additionally, protein, it is a popular part of low-calorie diets. the lack of standard guidelines for cheese In Bhutan, the local cottage cheese and butter production and grading leads to inconsistent supply are major dairy products and an important part of and inferior quality of cheese in the markets. the Bhutanese diet. It is also a major source of Moisture content determines the quality of protein. Local cottage cheese, popularly known as cheese. The international standards define cottage datshi, is produced using traditional processing cheese as the product containing moisture not 120 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 120-122, 2018 exceeding 80% and milk fat not less than 4% 3. RESULTS AND DISCUSSION (Chandhan 2003). According to the Prevention of 3.1 Moisture content in datshi Food Adulteration Act (PFA) 1976 and Codex Table 1 shows the average moisture content of standards for butter (Codex STAN 279-1971), the datshi from three districts. The moisture content moisture content of butter should not exceed 16%. ranged between 60.24% and 71.24% with overall However, no detail analysis of datshi and butter has average moisture content of 66.69%. The overall been conducted to assess their quality as well as average falls within the acceptable limits prescribed understand the compositional parameters of these (Indian standards and Codex General Standards) for dairy products in Bhutan. Therefore, a study was cheese. The Codex General standards for cheese conducted with the objectives to determine the (CODEX STAN 283-1978) states that moisture moisture content of datshi and butter available in content of more than 67% can be termed as soft the market and establish baseline data to facilitate cheese with a fat content of less than 10%, if systematic grading and standardization of quality of produced from skim milk while the Bureau of dairy products in the country. Indian standard (IS: 2785-1797) stipulates that the moisture content of soft cheese should be in the 2. MATERIALS AND METHOD range of 48–70% with a maximum of 13% milk fat 2.1 Study areas and sampling (for skim milk cheese). Based on the Codex The study was conducted in the districts of Standard and Indian Standard, most of the datshi Thimphu, Paro and Haa in western region of produced in three study districts can be classified Bhutan. Samples were randomly collected from under the soft type cheese or ripened/fresh cheese. Milk Processing Units (MPUs), dairy sales counters, retail shops, roadside vendors, and Table 1: Moisture content of datshi in three weekend vegetable markets in Thimphu, Paro and districts. Haa districts. Samples were collected from the District N Moisture content Standard month of April to June, 2017. Butter samples were % Deviation collected from only two districts of Thimphu and Paro. The samples were stored in a cool box with Haa 8 67.61 2.4 an average temperature of 20oC. The samples were Paro 9 66.25 3.2 then transported to the National Food Testing Thimphu 15 66.46 2.7 Laboratory of Bhutan Agriculture and Food Regulatory Authority (BAFRA), Yusipang, Thimphu, for analysis. The moisture content was 3.2 Moisture content of butter determined with the Oven Drying Method. The moisture content in butter samples ranged from 13.78% to 38.79% with an average moisture 2.2 Sample preparation for laboratory analysis content of 21.64%. The preliminary analysis of Datshi samples were manually broken into small moisture composition of butter showed a higher pieces and mixed properly, and a representative than acceptable level of moisture. Based on the sample of 2-3 gms was placed in an empty dish Prevention of Food Adulteration Act (PFA) 1976 and the Codex standards for butter (Codex STAN (W1) for the analysis. The dish with sample (W2) was weighed and recorded. Thereafter, the sample 279-1971), the upper limit of moisture content is was oven dried at 102°C for 2 hours (Official set at 16%, but in this study, the average moisture Methods of Analysis of AOAC International) till all content of butter samples was 21.64%. The high the free moisture was evaporated. The dried moisture content in butter reflects not only the poor samples were transferred to the desiccator for quality but would also mean that it will favor cooling. After cooling to room temperature, the microbial activity and reduce shelf life. Moisture rich foods are easily susceptible to microbial attack, dish with dried sample (W3) was weighed and recorded again. Water removed was considered as resulting in deterioration of the product. the weight loss on drying and accordingly, percent moisture was calculated using the following 4. CONCLUSION AND RECOMMENDATION formula. Based on the results, most of the datshi delivered to the consumers in Thimphu, Paro and Haa districts 푊 −푊 % 푚표푖푠푡푢푟푒 = 3 1 × 100 can be graded as soft cheese as per the moisture 푊2−푊1 content, however, the moisture content of butter is higher than the acceptable level. Therefore, there is Where, W1= Weight of empty dish, W2= Weight of a need to improve the quality of butter by reducing dish with fresh sample, and W3= Weight of dish moisture content. Similar study needs to be with dried sample. extended to other parts of Bhutan, to facilitate Yangchen et al. (2018) 121 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 120-122, 2018 development of compositional standards to ensure Upadhyay K (2003). Essentials of Cheesemaking. quality of cheese and butter. Anand: Anand Press, India. Robert L and Bradley J (2001). Determination of Acknowledgement moisture in cheese and cheese products. Journal We would like to sincerely acknowledge BAFRA of AOAC International, 12: 574-576. and the National Food Testing Laboratory, Livestock Statistics 2015. (2016). Thimphu: Yusipang, for analyzing the samples and allowing Department of Livestcok, MoAF, Thimphu, authors to conduct the study. The authors also Bhutan. thank Livestock Officers of Thimphu, Paro, Haa Google chrome. (n.d.). Retrieved March 10, 2017, and Program Director, NDRC, Yusipang, for from Google : Available at: support during the collection of samples. http://people.umass.edu/~mcclemen/581Moistu re.html, (Accessed on 21 January 2018). REFERENCES De S (1991). Outlines of Dairy Technology. Oxford University Press, Delhi, India. Codex General Standard for Cheese Codex Stan A- 6-1978, Rev.1-1999, Amended 2006. Indian standard, Specification for natural cheese (hard variety), processed cheese, processed cheese spread and soft cheese, IS: 2785 – 1979. Chandan RC (2003). Encyclopedia of Food Sciences and Nutrition (Second Edition). Available at: https://www.sciencedirect.com/topics/food- science/cottage-cheese. (accessed on May 13, 2017). Yangchen et al. (2018) 122 Bhutan Journal of Animal Science (BJAS) Volume 2, Issue 1, Page 123-126, 2018 Review paper Anti-Microbial Resistance (AMR) in Bhutanese livestock: a review NIRMAL K THAPA1*, PUSPA M SHARMA1, NARAPATI DAHAL2, SURYA B CHAMLING3, VIJAY RAIKA1, JAMBAY DORJEE4, RATNA B GURUNG1 AND KINZANG DUKPA1 1National Centre for Animal Health, Department of Livestock, Serbithang, Bhutan 2Animal Health Division, Department of Livestock, Thimphu, Bhutan 3National Poultry Research and Development Centre, Department of Livestock, Sarpang, Bhutan 4Regional Livestock Development Centre, Department of Livestock, Zhemgang, Bhutan *Author for correspondence: [email protected] Copyright © 2018 Nirmal K Thapa. The original work must be properly cited to permit unrestricted use, distribution, and reproduction of this article in any medium. ABSTRACT: The inappropriate use of antimicrobials in humans and livestock have led to development of resistance by bacterial pathogens. The studies on imported chicken carcass in 2007 revealed prevalence of Salmonella (13%) with Salmonella enteritidis serotype (84.62%) and Salmonella typhimurium (15.38%). The pathogens were found to be resistant to nalidixic acid, amoxicillin and cephalexin. Similarly, a study conducted in the domestically produced broiler chicken carcass during 2016 also detected Salmonella typhimurium and Salmonella paratyphimurium type B with prevalence of 73.9% and 26.1%, respectively. They were resistant to Tetracycline (95.6%), Trimethoprim (86.9%) and Amoxycillin (65.2%). The investigation of Escherichia coli in three government pig-breeding farms revealed (2.4%, 2/83) ESBL producers harbouring beta lactamase genes for CTX-M-15 and TEM-1, of which two isolates were multidrug resistant (MDR) and belonged to sequence type (ST) ST156 and ST4173, respectively. This indicates the emergence of MDR ESBL producing Escherichia coli among breeding pigs in Bhutan. Likewise, common pathogens like Escherichia coli (33%), Staph. Aureus (19%), Enterobacter (15%), Corynbacterium (11%), Streptococcous (8%), Pseudomonas (5%), Klebsiella pneumoniae (4%) were isolated from milk samples collected from dairy farms from various parts of the country. The pathogens were found resistant to antibiotics like Penicillin G (23%), Ampicillin (20%), Amoxicillin (20%), Erythromycin (17%), Tetracycline (7%), Gentamicin (7%) and Streptomycin (6%). Rational use of antibiotics must be promoted for controlling AMR. Keywords: Antibiotic; antimicrobial; pathogen; public health; risk. 123 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 123-126, 2018 1. INTRODUCTION procurement record were compiled by the Drugs Vaccines and Equipment Unit (DVEU) of Antimicrobials are a special class of medicines National Centre for Animal Health (NCAH). that have been long and widely used as medicine Annually, about 631 kg and 441 kg of in both humans and animals. They also contribute antimicrobials were procured during 2014-15 and to food security by meeting the increasing demand 2015-16, respectively. The antimicrobials are for food of animal origin. In animals, they are used supplied to the animal health facilities and central either for therapeutic and prophylactic purposes or farms throughout the country. Huge number of as growth promoters. However, its inappropriate oral antimicrobials are procured and distributed use over the past had led to emergence of bacterial compared to the injectable (Table 1). Table 2 resistance known as “Antimicrobial Resistance shows various antimicrobials under each (AMR)”. Globally, this has resulted in high classification used in the country. morbidity and mortality with serious social and economic implications in both humans and 3. Status of AMR in animals animals. Resistant pathogenic bacteria from Few studies revealed the presence of various humans, animals and environment can spread from bacterial pathogens, including the zoonotic ones one source to another and could pose a serious risk (Dahal 2007; Gurung 2016). The details of the to public health. The spread of these resistant pathogens are summarized in Table 3. A study organisms is through trade, travel and migration, conducted by Dahal (2007) on the prevalence of which has led to a global concern. Salmonella spp. and drug resistance in imported The World Organization for Animal Health chicken carcasses in Bhutan showed 13% (OIE), World Health Organization (WHO), Food prevalence of Salmonella with Salmonella and Agriculture Organization (FAO) are working enteritidis as the most frequently isolated serotype hand in hand to address the concern through the (84.62%), followed by Salmonella typhimurium tri-partite approach at a global and regional level. (15.38%). Among the seven antimicrobials tested, The OIE member states were recommended to resistance to nalidixic acid was the highest, pursue the same issue at the national level. In followed by amoxicillin and cephalexin. Another Bhutan, studies (Dahal 2007; Gurung 2016) have study conducted on domestically produced broiler revealed the existence of pathogens resistant to chicken carcass showed that the prevalence of various antibiotics. These pathogens detected were Salmonella typhimurium and Salmonella even zoonotic in nature. paratyphimurium type B were 73.9% and 26.1%, respectively. The antibiotic susceptibility test for 1. ANNUAL ANTIMICROBIAL CONSUMPTION 23 isolates of Salmonella species showed highest The annual consumption of antimicrobials was sensitivity to Gentamicin with 73.9%, followed by calculated based on the reporting option 3 of the Streptomycin (56.5%) and Ampicillin (47.8%). OIE template-over all amount sold or used in Similarly, the result showed the highest animals by antimicrobial class. This option was resistance to Tetracycline with 95.6%, followed by selected as the government of Bhutan provides Trimethoprim 86.9% and Amoxycillin 65.2% medicines free of cost. The data from the annual (Gurung 2016). The antibiotic susceptibility test Table 1: Quantities of antimicrobials consumed in the country (2013-2016) as per annual submission to OIE. 2014-15 2015-16 Class of Oral Inject. Others Total Oral Inject. Others Total antimicrobials (kg) (kg) (kg) (kg) (kg) (kg) (kg) (kg) Aminoglycosides 0 4 0 4 0 12 0 12 Penicillins 16 16 4 36 60 9 0 69 Cephalosporins 12 0 2 14 32 3 0 35 Fluoroquinolones 0 1.5 0 1.5 1 1 0 2 Sulfonamides 449 14 2 465 236 22 4 262 Tetracyclines 30 42 0 72 43 18 0 61 Others 0 40 0 40 0 0 0 0 Total 507 117 8 631 372 65 4 441 Thapa et al. (2018) 124 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 123-126, 2018 for 23 isolates of Salmonella species showed the antimicrobials have been reported in veterinary highest sensitivity to Gentamicin (73.9%), clinical practices, especially against mastitis followed by Streptomycin (56.5%), and (Chamling 2016). Ampicillin (47.8%). The investigation of Escherichia coli in three 4. SURVEILLANCE OF AMR IN BHUTAN government pig-breeding farms revealed (2.4%, Currently, the laboratory facility for Antibiotic 2/83) ESBL producers harbouring beta lactamase Sensitivity Testing (ABST) is available only at the genes for CTX-M-15 and TEM-1 (Gurung 2016). National Centre for Animal Health (NCAH) and the The two isolates were multidrug resistant (MDR) tests are conducted on case-by-case basis. and belonged to sequence type (ST) ST156 and Although, some of the laboratories of Regional ST4173, respectively. This indicates the emergence Livestock Development Centers (RLDCs) have of MDR ESBL producing E. coli among breeding facilities for ABST, they do not conduct the test. pigs in Bhutan and hence monitoring for ESBL producing bacteria should be continued in humans 5. LEGISLATION and animals in order to protect public health (Sharma et al. 2017). Antimicrobials for veterinary use is classified under In a study conducted on microbiological quality schedule E.2 prescription medicines (Medicine Act of raw milk in Bhutan (Chamling 2016), the of Kingdom of Bhutan 2003 and Bhutan Medicine common pathogens isolated were Escherichia coli Rules and Regulations 2012). As per this (33%), Staph. Aureus (19%), Enterobacter (15%), classification, the antimicrobials are allowed to be Corynbacterium (11%), Streptococcous (8%), sold or dispensed only with prescription. The Drug Pseudomonas (5%), Klebsiella pneumoniae (4%). Regulatory Authority (DRA) regulates the sale and The organisms were found resistant to antibiotics distribution of these medicines. In addition, DRA like Penicillin G (23%), Ampicillin (20%), also regulates the therapeutic use of antimicrobials Amoxycillin (20%), Erythromycin (17%), in animals, however, the use of antimicrobials as Tetracycline (7%), Gentamicin (7%) and feed additives/growth promoters are monitored by Streptomycin (6%). The susceptibility profile the Department of Livestock (DoL) with technical indicated Tetracycline (27%), Streptomycin (17%), advice of the National Veterinary Drug Committee Amoxycillin (17%), Erythromycin (16%), (NVDC). Ampicillin (14%), Penicillin G (6%) and Gentamicin (3%). Several treatment failures of 6. PREVENTION/CONTAINMENT ACTIVITIES Table 2: Antimicrobials used in the country as The antibiotic guidelines for animals in Bhutan per the classification. were developed under the funding support of WHO. DoL also developed the Standard Treatment Class Antimicrobials Guidelines for field staff, which helps in promoting Aminoglycosides Amikacin rational use of antimicrobials in the field. All Gentamicin implementing agencies are expected to adhere Cephalexin strictly to these guidelines and support to reduce development of antimicrobial resistance. Erythromycin Cephalosporins Amoxycillin REFERENCES Streptomycin Macrolides Cefotaxine MOH (2012). Bhutan Medicine Rules and Ceftriaxone Regulations 2012. Ministry of Health, Penicillins Ampicillin Thimphu, Bhutan. Cloxacillin Chamling S (2016) Proceedings of International Workshop on Development of Udder Health Penicillin G Control Programme in Bangladesh CVASU, Quinolones Tazobactum Chittagong, 31 May 2016: 45-55. Sulfonamides Sulfadimidine Dahal N. (2007). Prevalence and antimicrobial Trimethoprim resistance of Salmonella in imported chicken Sulfadiazine carcasses in Bhutan. MSc Thesis, Chiang Mai Sulfamethoxazole University and Freie Universitat Berlin, MSc Tetracyclines Tetracycline Program. Oxytetracycline Gurung M (2016). Prevalence of Salmonella in Others Metronidazole dressed broiler carcasses and their susceptibility Thapa et al. (2018) 125 Bhutan Journal of Animal Science (BJAS), Volume 2, Issue 1, Page 123-126, 2018 to antibiotics. BSc Thesis, College of Natural Sharma PM, Zurfluhb K, Nüesch-Inderbinenb, M, Resources, Lobesa, Punakha, Bhutan. Stephan R, Dukpa K and Gurung RB (2017). Marshall BM and Levy SB (2011). Food animals First Detection of Extended-Spectrum ß- and antimicrobials: Impacts on human health. Lactamase Producing Escherichia coli in Clinical Microbiological Review, 24: 718–733. Breeder Pigs in Bhutan. ARC Journal of MOH (2012). Medicine Act of Kingdom of Bhutan Animal and Veterinary Sciences, 3 (1): 13-17. 2003. Ministry of Health, Thimphu, Bhutan. Table 3: Pathogens with antibiotic resistance (antimicrobial resistance) profile. NA-Nalidixic acid, AMP- Ampicillin, TET- Tetracycline, AMOX-Amoxicillin, CIP-Ciprofloxacin, S- Streptomycin, TMP- Trimethoprim, GENT-Gentamycin, CF-Cephalothin, CTX-Cefotaxime, C-Chloramphenicol, SMZ- Sulphamethoxazole, K-Kanamycin, CE Cephalexin. Source of Pathogens Prevalence AMR profiles Investigator Pathogens (%) Chicken Salmonella Over all carcass (N=52 isolates) (13) (imported) S. entiridis 84.6 NAL (95.5%) AMO (13.6%) CE Dahal 2007 (n=44) (4.5%) CIP (2.3%) TMP (2.3%) S. typhymurium 15.4 NAL (100%) CE (12.5%) (n=8) Chicken Salmonella Over all carcass (n=23 isolates) (12.8) (Local) S. typhymurium 73.9 TET (95.6%), TMP (86.9%), AMO Gurung 2016 (n=17) (65.2%), AMP (47.8%), AMP (47.8%), GENT (13%), S (8.7%) S. paratyphi 26.1 type B (n=6) Faecal Escherichia samples (Pig coli breeding (n=83 isolates) farms) 2.4 AMP, CF, CTX, CIP, S, C, NA, Sharma et al. 2017 Extended- SMZ, TMP, TET, K Spectrum ß- Lactamase (ESBL) Producers Thapa et al. (2018) 126 BHUTAN JOURNAL OF ANIMAL SCIENCE (BJAS) INSTRUCTIONS FOR AUTHORS AIMS AND SCOPE The Bhutan Journal of Animal Science (BJAS), founded in 2016, is an English-language journal on livestock science. BJAS is supervised by the Department of Livestock, Ministry of Agriculture and Forests, and financed by the Royal Government of Bhutan. BJAS is published annually. BJAS publishes research, reviews, reports on all aspects of ruminants, poultry, aquaculture, and apiculture. BJAS also accepts livestock associated research reports on climate change, sustainable development, and rural livelihood. BJAS requires academic papers and research reports to display universal, strategic and innovative characteristics in both theory and practice. BJAS acts as a forum for sharing information between researchers, policy makers and practitioners. Research Article Criteria BJAS will consider papers describing original research findings related to ruminants, poultry, fishery, and forage sciences. Topics of papers can be on animal health, animal production, forage, and social sciences related to livestock. Research articles submitted to BJAS should be between 5,000 and 10,000 words, though longer submissions may be considered under exceptional circumstances. PREPARING YOUR MANUSCRIPT Please mention whether your submission is a research article, review, or short communication. Manuscripts must be written in concise English. Please use double line spacing. Include line and page numbering. The manuscript should be divided into the following sections: • Title page • Abstract, maximum 300 words • Keywords • Introduction • Study area • Methods • Results • Discussion • Conclusions • Declarations • References Title page The title page should: • present a title that best describes the content of the article. • list the full names and institutional addresses for all authors. If a collaboration group should be listed as an author, please list the group name as an author and include the names of the individual members of the group in the “Acknowledgements” section in accordance with the instructions below. Only those who have contributed to the concept and idea of the scientific article shall qualify as co-authors. Co-authors should appear in order of their contribution. • indicate the corresponding author with email address and phone numbers Abstract The abstract should briefly summarize the aim, findings or purpose of the article. Please minimize the use of abbreviations and do not cite references in the abstract. Keywords Three to eight keywords representing the main content of the article. 127 Main text This should contain the body of the article, and may also be broken into subsections with short, informative headings. List of abbreviations If abbreviations are used in the text they should be defined in the text at first use. Competing interests All financial and non-financial competing interests must be declared in this section. If you are unsure whether you or any of your co-authors have a competing interest, please contact the editorial board. Funding All sources of funding for the research reported should be declared. The role of the funding body in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript should be declared. Authors' contributions The individual contributions of authors to the manuscript should be specified in this section. Acknowledgements Please acknowledge anyone who contributed towards the article who does not meet the criteria for authorship including anyone who provided professional writing services or materials. Authors should obtain permission to acknowledge from all those mentioned in the Acknowledgements section. Authors' information You may choose to use this section to include any relevant information about the author(s) that may aid the reader's interpretation of the article and understand the standpoint of the author(s). This may include details about the authors' qualifications, current positions they hold at institutions or societies, or any other relevant background information. Please refer to authors using their initials. Note this section should not be used to describe any competing interests. How to format in-text citation and references Intext citation: Single author: Jigme Wangdi In-text citation: Wangdi (2017) / (Wangdi 2017) Two authors: Jigme Wangdi and Towchu Rabgay (2017) In-text citation: Wangdi and Rabgay (2017) / (Wangdi and Rabgay 2017) More than three authors: Jigme Wangdi, Towchu Rabgay, Namgay Dorji (2017) In-text citation: Wangdi et al. (2017) / (Wangdi et al. 2017) Reference style (example): Article within a journal Dorji T, and Tshering G. (1999). Side effects of excessive insemination in mithun bulls. Journal of Animal Science, 2 (Volume number): 12-15 (Page number). Complete book Gyaltshen T, Wangmo T, and Karpo K. (1985). World of Animals. Publisher, Place pf publication, country of publication. Book chapter, or an article within a book Chamling S. 1997. Crossbreeding of Yak and Buffalo (Chapter name). In Animal Reproduction (Name of book), Tshering L. and Timsina M. (editors), 99-119 (page number). Publisher’s name, place of publication. 128 Online document Gurung RB and Dukpa K. 2007. Name of the document. http://philsci- archive.pitt.edu/archive/00003390. Accessed 26 June 2007. File formats The word processor file format is acceptable for the main manuscript document Please note: Please do not submit the manuscript in pdf format. Figures must be submitted as separate image files (graphs in editable excel files and photographs in high resolution JPEG files) and not as part of the submitted manuscript file. Failing to submit these source files will cause unnecessary delays in the production process. Style and language For editors and reviewers to accurately assess the work presented in your manuscript you need to ensure the English language is of sufficient quality to be understood. If you need help with writing in English you should consider: • Asking a colleague who has a good command of language or preferably a native English speaker to review your manuscript for clarity. • Using a professional language editing service where editors will improve the English to ensure that your meaning is clear and identify problems that require your review. Please note that the use of a language editing service is not a requirement for publication and does not imply or guarantee that the article will be selected for peer review or accepted. Use of an editing service is neither a requirement nor a guarantee of acceptance for publication. Preparing figures When preparing figures, please follow the formatting instructions below. • Figures should be provided as separate files, not embedded in the main manuscript file. • Each figure of a manuscript should be submitted as a single file that fits on a single page in portrait format. • Multi-panel figures (those with parts a, b, c, d etc.) should be submitted as a single composite file that contains all parts of the figure. • Figures should be numbered in the order they are first mentioned in the text, and uploaded in this order. • Figures should be uploaded in the correct orientation. Figure file types We accept the following file formats for figures: • Microsoft Word (suitable for diagrams and/or images, figures must be a single page) • JPEG (suitable for photographic images, less suitable for graphical images) Figure size and resolution JPEG files saved at maximum quality should be submitted. Figures are resized during publication of the final full text and PDF versions. Preparing tables When preparing tables, please follow the formatting instructions below. • Tables should be numbered and cited in the text (i.e. Table 1, Table 2 etc.). • Tabular data provided as additional files can be uploaded as an Excel spreadsheet (.xls ) • Table titles (max 15 words) should be included above the table, and legends (max 300 words) should be included underneath the table. 129 Anti-Microbial Resistance (AMR) Antibiotics are essential resources for human health, animal health and animal welfare. Their misuse can result in the emergence of bacteria resistant to their action, leading to “Anti-Microbial Resistance” or “Anti-bioresistance”. This phenomenon deeply threatens the control of diseases worldwide. To preserve the effectiveness of antibiotics, collective efforts are needed to ensure responsible and prudent use of antibiotics in animals. WHAT CAN YOU DO AS Veterinarians or Animal health workers 1. Only prescribe and dispense antibiotics for animal under your care and only if necessary 2. Conduct antimicrobial sensitivity testing before prescribing or administering an antibiotic 3. Educate animal owners on the risks associated with misuse of antibiotics 4. Husbandry hygiene methods vaccination strategies, and, periodically review farm records to ensure compliance with your prescriptions 5. Keep your knowledge on antibiotic use recommendations up to date Contributor Dr. Nirmal K. Thapa Specialist National Center for Animal Health, Department of Livestock, Ministry of Agriculture and Forests, Thimphu, Bhutan PRIORITY SECTOR LENDING (PSL) GUIDELINES “TOWARDS PROMOTION OF COTTAGE AND SMALL-SCALE INDUSTRIES” CONTRIBUTED BY: Dr. Min Prasad Timsina