Improved productivity, profitability and sustainability of sheep production in Maharashtra, India through genetically enhanced prolificacy, growth and parasite resistance

Year 1 (01/01/2003-31/12/2003)
Production of appropriate genotypes for testing in shepherds' flocks (India)
This work is centred at the Nimbkar Agricultural Research Institute (NARI) where over 700 breeding ewes of various breeds and crosses are maintained. The breeding work at NARI has three broad purposes, namely:
the development of a fecund Deccani sheep by introgression of the FecB gene
the development of composite meat breed
the maintenance of sufficient Deccani purebreds to act as a benchmark for analysis of performance of the improved genotypes.
All breeding is done by artificial insemination (AI) using fresh semen to facilitate accurate pedigrees. Genotyping of lambs for the FecB gene occurs early in life using the PCR-RLFP test at the National Chemical Laboratory (NCL), Pune. By July-August 2003 there were 223 mature crossbred ewes carrying the FecB gene at NARI of which 197 were inseminated. This is slightly ahead of target. A total of 647 ewes were inseminated at this mating.

Testing of improved genotypes in shepherds' flocks and development of appropriate management technologies
A total of 78 ewes (38 FecBB+, 40 FecB++) were distributed amongst nine local shepherd flocks during 2003, exceeding the target for 2003. These ewes produced a total of 98 live lambs with 83 surviving lambs. Mortality did increase with litter size. In addition total of 10 FecBB+ (heterozygote) rams were introduced to 12 local flocks for 35-74 days producing 116 conceptions and 59 lambs. Of these lambs 29 have been shown to carry the FecB gene. Management practices of the participating shepherds are being closely monitored, particularly in relation to managing multiple births.
Regulation of expression of FecB in Indian and Australian sheep breeds (India and Australia)
In India, analysis of data at NARI revealed that one copy of FecB increased ovulation rate and litter size in multiparous ewes. The results were similar when records of ewes of different genetic backgrounds were added to the analysis, revealing that the FecB gene appears to act independently of the overall genetic background of carrier animals. The effect of the first copy of the FecB gene on ovulation rate in this study was lower than the 1.65 ova per copy reported for the Booroola Merino. However, the increase in litter size with one copy of the gene in this study is similar to that reported in the literature for the Booroola Merino.
An experiment examining the effect of nutrition on expression of the FecB trait in Garole ewes was conducted. The data still requires complete analysis but pre-mating level of feeding did not appear to have had a major effect on body weight or litter size possibly due to feed substitution effects.
In Australia, DNA samples have been collected from 428 Merino ewes on a commercial Merino property in Southern NSW into which the FecB gene had been introduced in the 1980s. Of these, 329 ewes have now been genotyped. When genotyping is complete the data will be analysed to determine the expression of the FecB trait in these genotypes and any associations with production variables. This represents the first large-scale genotyping of sheep for the FecB trait in Australia, using the PCR-RLFP test. The test is now established at UNE where most of this genotyping has occurred.
Development of extension and genetic models for the dissemination of proven genotypes (India and Australia)
In Australia and India a PhD student is nearing the end of the second year of study at UNE under an ACIAR John Allwright Fellow. Good progress has been made on developing economic values for sheep production traits in Maharashtran sheep production systems and modelling the impact of introgressing the FecB gene in these systems. This work will form an important part of her doctorial thesis, as will an analysis of methods of introgressing the gene into a large sheep population.

Objective A. Production of appropriate genotypes for testing in shepherds' flocks (India).
This work is centred at the Nimbkar Agricultural Research Institute (NARI) where over 600 breeding ewes of various breeds and crosses are maintained. The breeding work at NARI has three broad purposes, namely a) the development of a fecund Deccani sheep by introgression of the FecB gene; b) the development of a composite meat breed and c) the maintenance of sufficient Deccani purebreds to act as a benchmark for analysis of perfomance of the improved genotypes. All breeding is done by AI using fresh semen to facilitate accurate pedigrees. Genotyping of lambs for the FecB gene occurs early in life using the PCR-RFLP test at the National Cehmical Laboratory (NCL), Pune. During 2004 two major matings occurred; 232 ewes were inseminated in May 2004 giving rise to 228 lambs. In September-October 2004 a second breeding program was carried out with 341 ewes inseminated and 300 of these ewes in lamb at the end of the year. Of the 508 non-Garole ewes inseminated, 152 (30%) carried one copy of the FebB mutation while 356 ewes carried no copy of the mutation. The only 6 crossbred ewes homozygous for the FecB mutation were used in a MOET (multiple ovulation and embryo transfer program) between 12-15 July. Despite the transfer of 30 embryos to recipient ewes, no pregnancies resulted. This approach will not be tested again. A major cull of ewes was conducted during the year with 433 ewes culled. This assisted with managing the ongoing drought conditions in Maharashtra. EBVs for growth and reproductive traits have been estimated for all ewes and a selection index derived based on economic weights for variables of economic significance in Maharashtra. The index will be used to derive mating lists in early 2005.

At the National Chemical Laboratory in Pune, a total of 529 DNA samples from NARI flock sheep were genotyped for the FecB mutation using PCR-RFLP. Studies into the genetic diversity of the Garole sheep at NARI, using microsatellite markers were able to demonstrate relatively good genetic diversity.

Objective B. Testing of improved genotypes in shepherds' flocks and development of appropriate management technologies.
In 2003 the late distribution of pregnant 'Fecund Deccani' ewes carrying the FecB gene to shepherds' flocks during drought conditions led to very high lamb mortality of (52%). These ewes have now lambed a second time with lamb mortality to 6 months of age of only 7.7%. Twenty-nine FecB ewes reared 38 lambs to 3 months of age (131%) compared with 23 lambs reared by 25 FecB ewes (92%). This suggests that once adapted to shepherd flock conditions, FecB ewes can significantly life reproductive rates and profitability. Shepherds are now happy with these animals and their performance.

During 2004, 21 FecB rams were also used to mate shepherds' ewes in 18 selected flocks. This produced 255 live lambs with an additional 63 lambs born from ewes mated to the late 2003 distribution of rams. From the 318 lambs produced, 60 female lambs are carriers of the FecB mutation. In addition, out of 147 lambs born from oestrus synchronization and artificial insemination programs there are 61 ewe lambs that are carriers of the FecB mutation. At the National Chemical Laboratory in Pune a total of 379 DNA samples from sheep from 21 participating shepherds' flocks were genotyped for the FecB mutation using PCR-RFLP.

During November 2004, 32 pregnant 'NARI composite' ewes (12 FecB B+ and 20 Fec B ++) as well as 10 more pregnant Fecund Deccani types (FecB B+) were distributed to 6 shepherds. The distribution program to shepherds' flocks is well in excess of what is stated in the project document, with involvement of 26 shepherds' flocks (4 discontinued). The next year will be a crucial year for estimating the productivity of adapted ewes carrying the FecB mutation in shepherds' flocks. Shepherds' procedures for managing multiple births have been closely observed and documented during 2004 and a number of new strategies devised for implementation in 2005.

At NCL, 412 DNA samples collected from the Garole breed (n=20 each; Lonand, Sangola, Kolhapuri, Madgyal, Sangamneri) the Bannur (n=26) and the Madras Red )from Tamil Nadu, n=20) were analysed for the FecB allele. It was shown only to be present in the Garole breed. Thus it is unlikely that the gene pre-exists in flocks into which it is being introduced from NARI.

Objective C. Regulation of expression of FecB in Indian and Australian breeds of sheep (India and Australia).
In India, breeding values for litter size were estimated for all Garole ewes and rams and the flock broken into two groups, a low EBV group and a higher EbV group. The low EBV group will be mated to low EBV rams each year in an attempt to diverge from a 'Control Line'. This will be established from the high EBV group by mating to rams selected at random. The aim is to test whether there is a genetic basis for differences in expression in the FecB trait. Blood samples from 10 Garole ewes and 5 rams having extreme values for litter size EBV have been supplied by NARI to NCL for genetic analysis. At NCL 8 pairs of primers have been designed to span the whole DNA sequence of the BMP-IB gene which is the site of the FecB mutation. This will enable detection of mutations other than the FecB mutation, or variations of it, which may be responsible for variation in expression of the FecB trait. Alternatively variation may be due to loci in the region flanking the gene and scientists at NCL have designed PCR assays for 4 microsatellite markers closely flankiing the gene (JL26, JL2, JL36, JP27).

In Australia, a total of 460 ewes have been genotyped for the FecB allele from 'Allandale' a commercial Merino property in Southern NSW into which the FecB gene had been introduced in the 1980s. Of these, 68 are FecB BB, 204 are FecB B+ and 188 are FecB ++. The FecB allele does not appear to confer any disadvantage in productivity to animals carrying it, with the possible exception of a non-significant trend towards lower liveweight. This will necessitate a change in Objective C4. From the UNE Booroola flock, all 13 rams and 54 ewes tested were FecB BB confirming the homozygous nature of the flock.

The comparative reproductive endocrinology of FecB carriers of different breeds is being investigated at UNE. Oestradiol implants have been shown to significantly reduce ovulation rate (from 6.5 to 5.0) in FecB BB ewes with the probable mechanism of action being reduced FSH levels. Plasma samples from 5 FecB BB and 5 FecB B+ Garole ewes taken throughout the oestrus cycle have also been sent to UNE and assayed for a range of reproductive and metabolic hormones. These data await analysis.

Objective D. Development of extension and genetic models for the dissemination of proven genotypes (India and Australia).
This objective will be addressed during the last year of the project. There is considerable extension activity occurring in India with the collaborating shepherds.

Objective A. Production of appropriate genotypes for testing in shepherds' flocks (India).
The breeding program at NARI is now based on a selection index based on estimated breeding values and
economic values for 4 traits namely three-month weight, fertility, litter size and lamb survival. Using the index
some 25% of mature ewes were culled during the year (161/660). Matings for 2006 will be designed using the
TGRM software.

In Feb-March 2005 297 lambs were born at NARI from an AI program in Sept-October 2004. From 94 FecBB+
ewes inseminated, 102 live lambs were produced (109% lambing) and 96 lambs reached 3 months of age (102%
weaning). From 247 FecB++ ewes inseminated, 195 live lambs were produced (79% lambing) and 190 lambs
reached 3 months of age (77% weaning). Overall lamb mortality to 3 months was 3.7% which is very low.
Two further AI programs took place at NARI during 1995. In Feb-March 2005 213 ewes were inseminated
producing 190 lambs born and 172 lambs reared to 3 months. Overall lamb mortality of 9% was moderate.
Lambing % for FecBBB (n=4), FecBB+ (n=93) and FecB++ (n=116), ewes respectively was 100%, 100% and 80%.
Weaning % (to 3 months of age) was 100%, 88% and 75% respectively. A further 258 ewes were inseminated in
September October and these will lamb in Feb 2006.
A total of 487 lambs was born during the year of which 70 were FecBBB, 315 were FecBB+ 102 were FecB++. It is
pleasing that 79% of all lambs born contained the FecB mutation. Lamb mortality to 6 months of age was a
relatively high 20% due to an outbreak of bluetongue disease in the district. By comparison mortality in 2004 was
9%.

Total sheep numbers at NARI at the end of 2005 were 845. Amongst the crossbred ewe population there are 14
FecBBB, 111 FecBB+ and 93 FecB++ ewes available for breeding in February 2006.
During the year 354 DNA samples from NARI and 155 from shepherds' flocks were genotyped for the FecB
mutation at NCL.
Objective B: Testing of improved genotypes in shepherds' flocks and development of appropriate
management technologies.

To date 60 FecBBB+ and 60 FecBB++ ewes have been distributed to 22 smallholders from NARI. In addition 20 AI
programs using semen from rams carrying the FecB gene have been carried out and multiple ram introductions
made using NARI rams carrying the FecB mutation. Currently there are 21 smallholders with 1200 breeding ewes
involved in this objective. Data from the 2004 and 2005 lambings of distributed ewes indicate that one copy of the
FecB mutation confers an additional 0.3 lamb per ewe born and 0.14 additional lambs reared to 6 months of age.

Objective A: Ongoing production of appropriate genotypes for testing in shepherds' flocks
During 2006, two AI programs were carried out in the NARI sheep flock; 531 ewes were inseminated, 356 ewes lambed, 486 lambs were born and 421 were weaned. The average litter size of the NARI flock increased from 1.2 in February to 1.5 in September 2006. Twenty six rams were used in the first program; nineteen of these and an additional fifteen young rams were used in the second program. The primary selection of rams and ewes was done using a selection index of EBVs for reproduction and growth traits. Conformation and facial features preferred by local shepherds were also considered as selection criteria. Allotment of rams to ewes (mate selection) was done using the TGRM program to maximize genetic merit while minimizing relationships in the flock. During the year, 263 DNA samples from NARI and 107 from shepherds' flocks were genotyped by NARI and NCL staff members jointly.

All lambing records at NARI since 1996 were analyzed (3912 records of FecB+/FecB+ ewes, 1167 of FecBB/FecB+ ewes and 37 of FecBB/FecBB ewes). The number of lambs born alive and weaned per ewe inseminated (and per ewe lambed) was estimated to be 0.97 and 0.90 (1.02 and 0.95) for FecB+/FecB+ ewes, 1.43 and 1.24 (1.56 and 1.35) for FecBB/FecB+ ewes and 1.63 and 1.33 (1.72 and 1.40) for FecBB/FecBB ewes. At the end of 2006 there are 510 ewes available for breeding. For the first time, more of these (i.e. 270) are FecBB/FecB+ ewes than FecB+/FecB+ ewes (200) and there are 40 FecBB/FecBB ewes.

A nucleus Garole flock of 58 ewes and 40 rams is being maintained at NARI. Sixty seven new lambs were born during December 2006 and 57 of these (24 males and 33 females) were alive at the end of 2006. The Garoles have now adapted well to conditions at NARI.

Objective B: Ongoing dissemination of improved FecB carrier rams and semen into shepherds' flocks and evaluation of performance data
Ten homozygous and two heterozygous FecB carrier rams were introduced into eight shepherd flocks for breeding. Oestrus synchronization and artificial insemination were carried out using homozygous FecB carrier ram semen in nine flocks. These flocks with 818 ewes in total are intensively monitored. Additionally, 11 FecB carrier rams were introduced into 11 'less intensively monitored' flocks with 442 breeding ewes in total. NARI's rams have sired total 260 lambs in these flocks during the year, lambing is in progress in six flocks and will commence in the next two months in six flocks. The proportion of FecB carrier adult breeding ewes in intensively monitored participating flocks has increased from 13% to 20% over the reporting period. The number of lambs born alive and weaned per ewe lambing was 1.02 and 0.92 for FecB+/FecB+ ewes and 1.48 and 1.09 for FecBB/FecB+ ewes in 16 flocks. The figures for 10 of these flocks which were better managed were similar for FecB+/FecB+ ewes but for FecBB/FecB+ ewes, they were 1.55 lambs born and 1.26 lambs weaned per ewe lambing.

Four workshops were conducted at NARI's Lundy farm for dissemination of project results with about 25 shepherds participating in each one. The workshops were organized when there were 2-3 month old lambs in the NARI flock to show to the shepherds how the ewes and lambs are managed at NARI.

NARI organized a workshop on "Harnessing genetics to increase productivity of sheep and Bluetongue disease of sheep" on 8 November 2006. Around 500 shepherds attended. Ms. Leena Mehendale, Principal Secretary of the Animal Husbandry Department of Government of Maharashtra inaugurated the workshop.

A meeting of 60 government animal husbandry officers from the 32 districts of Maharashtra State organized in Pune by the Commissioner, Animal Husbandry was addressed by Dr. Pradip Ghalsasi. He informed them about the effect of the FecB gene on the reproductive performance of the two new strains of sheep being developed at NARI and the proper way to manage carrier ewes with a higher twinning percentage than the Deccani.

Objective C: Socio-economic analysis of the FecB dissemination program implemented in local smallholder flocks from 2003-06
A draft survey questionnaire was prepared in September 2006 in consultation with two agricultural extension specialists, Mr. Julian Prior from the University of New England, Australia and Dr. D. V. Rangnekar from Ahmedabad, India for interviewing the shepherd households where FecB carrier rams/semen and/or ewes were introduced. The questionnaire was modified by NARI researchers, translated into Marathi and nine of the participating shepherds were interviewed from November 2006 to January 2007. Shepherds' general perception is that they can get one and a half times as much income if a ewe has twin lambs rather than a single. They realize, however, that extra feed has to be given to twin-bearing ewes and to twin-born lambs.

Objective A: Ongoing production of appropriate genotypes for testing in shepherds' flocks
During 2007, two AI programs were carried out in the NARI sheep flock; 536 ewes were inseminated, 352 ewes lambed, 461 lambs were born and 423 were weaned. Large numbers of breeding rams were used by artificial insemination (27 and 43 in the two programs) to control inbreeding. Rams and ewes were selected primarily using a selection index of EBVs for reproduction and growth traits. Conformation and facial features preferred by local shepherds were also used as selection criteria. Ramms were allotted to ewes using the TGRM program to maximize genetic merit while minimizing relationships in the flock. During the year, 342 DNA samples from NARI and 313 from shepherds' flocks were genotyped by NARI and NCL staff members jointly. We have succeeded in reducing the proportion of non-amplified or doubtful samples that had to be repeat tested to 12% and 28% (from 50% in 2006) among NARI and shepherds' animals respectively.
Lambing records at NARI from 2002 to 2007 were analyzed as these were the years with substantial records of heterozygous and homozygous FecB carrier ewes. For 808 non-carrier, 532 heterozygous and 47 homozygous ewes conception rates to artificial insemination were 74%, 80% and 76% respectively. For these same groups, litter sizes at birth per ewe lambed were 1.02, 1,57 and 1.63; and at 3 months 0.96, 1.33 and 1.30.
These data indicate that litter size in homozygous ewes are very similar to heterozygoous ewes. This is an important new result which suggests that the risk of adverse reproductive outcomes in homozygous ewes due to excessive litter size is low.
At the end of 2007 there are 500 ewes available for breeding. Out of these, 80 are FecB homozygous, 260 are heterozygous and 160 are non-carrier ewes. A nucleus Garole flock of 46 adult ewes, 27 young ewes and 7 adult rams is being maintained at NARI.
Objective B: Ongoing dissemination of improved FecB carrier rams and semen into shepherds' flocks and evaluation of performance data
Eighteen homozygous FecB carrier rams were introduced into 13 shepherd flocks for breeding. These flocks with about 900 ewes in total are intensively monitored i.e. All animals have ear tags, are weighed every two months and lambs are genotyped. Additionally, 18 homozygous and 8 heterozygous FecB carrier rams were introduced into 19 'less intensively monitored' flocks with about 1000 breeding ewes in total. Animals in these flocks are not ear tagged; nor weighed and only female lambs are genotyped from those flocks where the breeding rams are heterozygous for FecB. NARI's rams have sired total 287 lambs in these flocks during the year and NARI's ewes in these flocks gave birth to 52 lambs during the year. The number of lambs is low because some rams had to be withdrawn due to a bluetongue outbreak and by the time they could be reintroduced, the ewes in those flocks were pregnant. There is also always a conflict when a significant number of rams has to be kept behind for NARI's own breeding program. The proportion of FecB carrier adult breeding ewes in intensively monitored participating flocks has increased from 20% to 28% over the reporting period (245 out of 876). A table is given in the appendix with the number of one year old ewes in all 16 intensively monitored flocks.
Data files are being prepared for final analysis of biological and economic benefits from introduction of the FecB gene. We hope to finish this analysis by the end of June 2008.
In 4 FecB homozygous, 196 heterozygous and 1360 non-carrier ewes in participating shepherds flocks the Litter sizes at birth per ewe lambed were 1.02, 1.44, 1.69.
Ten training workshops were conducted for shepherds during the year. In total, 644 men and women shepherds availed of these. One was a short training course on 'First aid treatment in sheep' for 62 shepherd men and women. All participants were given a first-aid kit and a simple Marathi booklet on first-aid in sheep. Out of the 9 other programs, 2 were held at NARI's Lundy farm and 4 outside Phaltan by NARI while 3 were organized by State government agencies and other NGOs. In these programs, the concepts that twinning can be introduced in sheep and can increase profits were explained to the participating shepherds. They were shown the CD prepared by NARI about the management of twin lambs and their questions were answered. About ten of the participants later visited NARI's Wadjal farm to see the NARI Suwarna breeding rams.
Objective C: Socio-economic analysis of the FecB dissemination program implemented in local smallholder flocks from 2003-07
Interviews of 18 shepherds participating in the project, whose flocks are intensively monitored, were held from November 2006 to February 2007. Eight shepherds with whom we had to stop working due to various reasons, were interviewed in August 2007. The draft survey questionnaire prepared in 2006 was used for this. This questionnaire was re-examined in 2007 in the light of the filled questionnaires to see whether it achieved the objectives of the survey. We modified it based on the experience gained and it was used to conduct a second round of interviews of 15 of the same shepherds in April 2008, as decided in the last coordination meeting. A marked change in management brought in by all the shepherds is that they have started keeping the lambs back when the ewes go grazing and giving them nutritious leguminous fodder grown by them. They have all seen a beneficial effect of this practice on lamb weights and price gained.