UNIVERSIDADE ESTADUAL PAULISTA – UNESP CAMPUS JABOTICABAL GENETIC CHARACTERIZATION OF RESISTANCE TO Haemonchus contortus IN MORADA NOVA SHEEP Marei Borsch von Haehling Veterinarian 2020 UNIVERSIDADE ESTADUAL PAULISTA – UNESP CAMPUS JABOTICABAL GENETIC CHARACTERIZATION OF RESISTANCE TO Haemonchus contortus IN MORADA NOVA SHEEP Marei Borsch von Haehling Supervisor: Dr. (PhD) Ana Carolina de Souza Chagas Thesis presented to the faculty of Agricultural and Veterinary Sciences – Unesp, campus of Jaboticabal, as part of the requirements for the acquisition of the title “Mestre” in Veterinary Medicine (area: animal pathology) 2020 CURRICULUM VITAE Marei Borsch von Haehling (née Borsch) was born in Cologne, Germany, on November 25th 1986. From 2007 to 2013, she studied veterinary medicine at the Ludwig– Maximilians–University Munich, obtaining the veterinary licence in May 2013. From 2009 to 2011, she worked as a graduate assistant in the department for veterinary anatomy. Internships at the veterinary practice Dr. Morsy in Wershofen (March 2010), as part of the clinical rotation at the clinics of the Ludwig–Maximilians–University for ruminants, equines, swine, birds and small animals (March 2011 to March 2012), at the veterinary practice Dr. Miguel Arcanjo Valencise in Dourado, Brazil (May 2012), at the clinic for equines in Kottenforst (June to July, 2012), at the department for animal welfare, ethology, animal hygiene and husbandry of the Ludwig–Maximilians–University (July 2012) and at the clinic for swine and small ruminants of the University of Veterinary Medicine in Hannover (July to September 2012) were completed successfully. She worked as a veterinarian at the veterinary practice Dr. Schneider and Gilg in Eichstätt (June 2013 to May 2014) and Dr. Zauscher in Odelzhausen (June 2014 to November 2016). In March 2018, she joined the master’s programme of the FCAV – Unesp of Jaboticabal, with execution of the experiments at the Embrapa Southeast Livestock Unit in São Carlos, SP, Brazil. AGRADECIMENTOS (ACKNOWLEDGEMENTS) Ao meu marido Ricardo, à minha sogra Calau, à minha cunhada Veri, à Mariana e à Sandi. Em cada minuto que eu me dediquei ao trabalho, eu sabia que vocês se dedicaram à minha filha, com muito amor e carinho. À minha orientadora, Dra Ana Carolina de Souza Chagas, pela oportunidade e pelo apoio nos momentos difíceis. À Dra Simone Meio Niciura, pelos ensinamentos no laboratório, na genética molecular e na arte de escrever um artigo, pelo sorriso e pela disposição extraordinária. À Dra Patrícia Tholon, pela introdução na estatística e análise genética e por ter insistido no “R”. Ao Dr. Waldomiro Barióni Júnior, pela ajuda na estatística e pelas histórias. À Giovanna, à Carolzinha, ao Giba e à Flávia, pelo apoio no laboratório. Ao Dr. Sérgio de Novita Esteves e aos funcionários de campo da Embrapa Pecuária Sudeste, pela manutenção dos animais e pela ajuda nas colheitas. À equipe do laboratório de parasitologia, Isabella, Amanda, Luciana, Alemán, João, Júnior, Leo e Rafa, pela grande ajuda e pela companhia nas colheitas. i SUMMARY page FIGURES................................................................................................................................ii TABLES.................................................................................................................................iii CERTIFICATE OF THE ETHICS COMMITTEE FOR ANIMAL EXPERIMENTATION...........v ABSTRACT............................................................................................................................vi RESUMO..............................................................................................................................vii CHAPTER 1 – PRELIMINARY CONSIDERATIONS.............................................................1 1.1. INTRODUCTION.............................................................................................................1 1.2. LITERATURE REVIEW...................................................................................................2 1.2.1. Relevance of gastrointestinal nematodes....................................................................2 1.2.2. Characteristics and potential of the Morada Nova breed............................................4 1.2.3. The host’s response to parasites.................................................................................6 1.2.4. Epidemiological factors of resistance to GIN...............................................................8 1.2.5. Basic concepts of animal breeding..............................................................................9 1.2.6. Selection of animals resistant to GIN infection............................................................9 1.2.7. Selection of animals resilient to GIN infection...........................................................11 1.2.8. Genetic correlations between resistance and performance......................................13 1.2.9. Variation between breeds and genotype by environment interactions......................14 1.2.10. Genetic markers.......................................................................................................15 1.2.11. Genetic markers for resistance to GIN in sheep......................................................16 1.3. REFERENCES..............................................................................................................17 CHAPTER 2 – GENETIC PARAMETERS FOR FAECAL EGG COUNT (FEC) PACKED CELL VOLUME (PCV) AND DAILY WEIGHT GAIN IN MORADA NOVA SHEEP INFECTED WITH Haemonchus contortus..............................................................................................24 2.1. ABSTRACT...................................................................................................................24 2.2. INTRODUCTION...........................................................................................................25 2.3. MATERIAL AND METHODS.........................................................................................26 2.3.1. Experimental animals and phenotypes......................................................................26 2.3.2. Statistical analysis......................................................................................................28 2.4. RESULTS......................................................................................................................30 2.5. DISCUSSION................................................................................................................37 2.6. CONCLUSION..............................................................................................................41 2.7. REFERENCES..............................................................................................................41 CHAPTER 3 – FOUR SINGLE NUCLEOTIDE POLYMORPHISMS (SNPs) ARE ASSOCIATED WITH RESISTANCE AND RESILIENCE TO Haemonchus contortus IN BRAZILIAN MORADA NOVA SHEEP..................................................................................45 3.1. ABSTRACT...................................................................................................................45 3.2. INTRODUCTION...........................................................................................................46 3.3. MATERIAL AND METHODS.........................................................................................48 3.3.1. Experimental animals and phenotypes......................................................................48 3.3.2. Genotypes..................................................................................................................48 3.3.3. Statistical analysis......................................................................................................49 3.3.4. Annotation and functional analysis of SNPs..............................................................50 3.4. RESULTS......................................................................................................................50 3.5. DISCUSSION................................................................................................................59 3.6. CONCLUSION..............................................................................................................63 REFERENCES.....................................................................................................................63 CHAPTER 4 – FINAL CONSIDERATIONS.........................................................................68 APPENDIX A........................................................................................................................70 APPENDIX B........................................................................................................................72 ii FIGURES page Figure 2.1. Frequencies of lambs sired by the rams 2014 and 287 in groups formed according to estimated breeding values (EBVs) of overall FEC (A), overall PCV (B) and total weight gain (C), with group 10 representing the most favourable, and group 1 the most unfavourable EBVs ……………………………………………………………………………….36 Figure 3.1. Box-and-whisker plot of genotypic effects of polymorphic SNPs (OAR2_14765360 (OAR2), OAR6_81718546 (OAR6), OAR11_62887032 (OAR11) and OAR12_69606944 (OAR12)) on mean faecal egg counts (FEC) in the first (meanFEC1) and second (meanFEC2) challenges, and total (totalmeanFEC), detected in 256 Morada Nova lambs …………..…………………………………………………………..……………….52 Figure 3.2. Box-and-whisker plot of genotypic effects of polymorphic SNPS (OAR2_14765360 (OAR2) and OAR12_69606944 (OAR12)) on faecal egg counts (FEC) of individual sampling dates (days 21, 28, 35 and 42 of the challenge 2), detected in 256 Morada Nova lambs …………………………………………...……………...…………………53 Figure 3.3. Box-and-whisker plot of genotypic effects of polymorphic SNPs (OAR2_14765360 (OAR2), OAR6_81718546 (OAR6), OAR11_62887032 (OAR11) and OAR12_69606944 (OAR12)) on mean packed cell volume (PCV) in the first (meanPCV1) and second (meanPCV2) challenges, and total (totalmeanPCV), detected in 256 Morada Nova lambs ……………………………………………………………………………………….54 Figure 3.4. Box-and-whisker plot of genotypic effects of polymorphic SNPs (OAR6_81718546 (OAR6) and OAR12_69606944 (OAR12)) on packed cell volume (PCV) of individual sampling dates (days 14, 28 and 42 of the challenge 2), detected in 256 Morada Nova lambs………………………………………………………………………………55 Figure 3.5. Box-and-whisker plot of genotypic effects of polymorphic SNPs (OAR2_14765360 (OAR2), OAR6_81718546 (OAR6), OAR11_62887032 (OAR11) and OAR12_69606944 (OAR12)) on daily weight gain (DWG) in the first (DWG1) and second (DWG2) challenges, and total (totalDWG), detected in 256 Morada Nova lambs ………..56 Figure 3.6. Box-and-whisker plot of genotypic effects of polymorphic SNPs (OAR6_81718546 (OAR6), OAR11_62887032 (OAR11) and OAR12_69606944 (OAR12)) on body weight at weaning and at individual sampling dates (day 42 in challenges 1 and 2), detected in 256 Morada Nova lambs ………………………………………………………57 iii TABLES page Table 2.1. Descriptive statistics for faecal egg count (FEC), packed cell volume (PCV), body weight (BW) and daily weight gain (DWG) in Morada Nova lambs.…….…………….31 Table 2.2. Descriptive statistics for faecal egg count (FEC) and packed cell volume (PCV) in Morada Nova ewes..…...…...………..………..……….…..……………….…...…………...32 Table 2.3. Estimates (± standard error) of additive genetic variance (σ2 a), residual variance (σ2 e), permanent environmental variance (σ2 pe), heritability (h2) and repeatability (t) for faecal egg count (FEC), packed cell volume (PCV), body weight (BW) and daily weight gain (DWG) of Morada Nova lambs……..…..…………………………………………….………..33 Table 2.4. Estimates (± standard error) of additive genetic variance (σ2 a), residual variance (σ2 e), permanent environmental variance (σ2 pe), heritability (h2) and repeatability (t) for faecal egg count (FEC) and packed cell volume (PCV) in Morada Nova ewes ………………………………………………………………...……..……………………………..34 Table 2.5. Phenotypic (rp) and genetic (rg) correlations (± standard error) at weaning, for overall traits, per challenge and on individual sampling dates, according to phenotypic values and estimated breeding values (EBVs), between the traits faecal egg count (FEC) and body weight (BW) or daily weight gain (DWG), packed cell volume (PCV) and BW or DWG, and FEC and PCV in Morada Nova lambs ……….…………………………………...35 Table 3.1. Genotypic and allelic frequencies of OAR2_14765360 (OAR2), OAR6_81718546 (OAR6), OAR11_62887032 (OAR11) and OAR12_69606944 (OAR12) SNPs in Morada Nova ewes and lambs ……………………………………………………….51 Table 3.2. Number of animals with OAR2_14765360 and OAR12_69606944 genotypes in the 20% lambs with most favourable and 20% of lambs with most unfavourable phenotypes for FEC (low and high FEC, respectively) ….…………………………………...58 Table 3.3. Number of animals with OAR6_81718546 and OAR12_69606944 genotypes in the 20% lambs with most favourable and 20% of lambs with most unfavourable phenotypes for PCV (high and low PCV, respectively) ………………………………………58 Table 3.4. Number of animals with OAR6_81718546, OAR11_62887032 and OAR12_69606944 genotypes in the 20% lambs with most favourable and 20% of lambs with most unfavourable phenotypes for daily weight gain (high and low DWG, respectively) ………………………………………………………………………………………………………59 Table A.1. Phenotypic correlations (± standard error) within traits for faecal egg count (FEC), packed cell volume (PCV), body weight and daily weight gain (DWG) in Morada nova lambs……………..………………………………………………………………………….70 iv Table A.2. Genetic correlations (± standard error) within traits for faecal egg count (FEC), packed cell volume (PCV), body weight and daily weight gain (DWG) based on estimated breeding values (EBVs) of Morada nova lambs ………………………………………………71 Table B.1. SNP positions and genes (name, type, distance to the SNP and function) located within a 1,000 kb window around each SNP ………………………………………...72 v CERTIFICATE OF THE ETHICS COMMITTEE FOR ANIMAL EXPERIMENTATION vi GENETIC CHARACTERIZATION OF RESISTANCE TO Haemonchus contortus IN MORADA NOVA SHEEP ABSTRACT – Gastrointestinal nematodes are a major constraint in sheep production. Breeding for resistance has proven to be an effective and feasible approach to address this problem. The use and investigation of genetic markers for resistance traits could accelerate genetic progress and lead to a better understanding of underlying molecular mechanisms. The aim of this study was to verify the possibility of selection for resistance and resilience traits in Morada Nova lambs, to estimate potential correlated responses and to evaluate if five single nucleotide polymorphisms (SNPs) (OAR2_14765360, OAR6_81718546, OAR11_62887032, OAR12_69606944 and OAR15_59871543) are associated with resistance and resilience traits. A total of 287 lambs and 131 ewes were submitted to two consecutive independent parasite challenges by oral infection with 4,000 infective larvae (L3) of Haemonchus contortus. Faecal egg counts (FEC), packed cell volume (PVC) and body weight were measured every one or two weeks for 42 days in each trial. DNA samples from 287 lambs, 131 ewes and 4 rams were amplified by ARMS-PCR or PCR-RFLP and genotypes were determined. Estimates of genetic parameters were obtained for individual records as well as for overall traits with repeated measures, using mixed animal models. Analysis of variance (ANOVA) was used for association analyses between SNP genotypes and phenotypes. In case of significant association, the allele substitution effect was calculated based on a linear model. Heritability estimates of FEC in the first and second parasite challenge were, respectively, 0.25 ± 0.18 and 0.46 ± 0.19 for lambs, and 0.00 ± 0.09 and 0.20 ± 0.16 for ewes. For PCV, heritability estimates were 0.23 ± 0.14 and 0.32 ± 0.16 for lambs and 0.13 ± 0.11 and 0.37 ± 0.18 for ewes. The overall weight gain heritability estimate was 0.70 ± 0.21. Genetic correlations of FEC and PCV between lambs and ewes were 0.36 ± 0.08 and 0.42 ± 0.08, respectively. No significant genetic correlation was found between weight gain and the other traits, while there was a negative genetic correlation between FEC and PCV (-0.70 ± 0.03). OAR2_14765360 and OAR12_69606944 were associated with FEC, and OAR12_69606944 also had significant effects on PCV and weight gain, showing favourable associations of the CC genotype with all evaluated traits. Both OAR6_81718546 and OAR11_62887032 were associated with weight gain, and OAR6_81718546 had an additional effect on PCV. OAR15_59871543 was not polymorphic in the population. OAR6_81718546 and OAR12_69606944 presented significant allele substitution effects of -1.06 ± 0.52 kg for the T allele on final body weight and 0.74 ± 0.32 for the C allele in PCV of the same sampling date, respectively. Selection for low FEC and high PCV is possible in Morada Nova lambs. Selection for low FEC should have a correlated response on PCV (leading to higher PCV), while no correlated response is expected on weight gain. Selection for low FEC and high PCV in lambs should lead to low FEC and high PCV in ewes. An index consisting of overall weight gain and two records of FEC and PCV (taken 4 to 6 weeks after infection during the second parasite challenge) can be used as selection criterion in the Morada Nova lambs, allowing simultaneous selection for lower FEC, higher PCV and higher weight gain. Our findings support the existence of quantitative trait loci (QTL) for resistance and resilience in linkage disequilibrium with the polymorphic SNPs and suggest their future use for explorations of these traits in Morada Nova sheep. Keywords: genetic markers, genetic parameters, host resistance, resilience, parasite challenge vii CARACTERIZAÇÃO GENÉTICA DA RESISTÊNCIA A Haemonchus contortus EM OVINOS DA RAÇA MORADA NOVA RESUMO – Nematódeos gastrintestinais são um grande obstáculo na produção de ovinos. A seleção para resistência tem sido uma abordagem eficiente e factível para lidar com esse problema. A utilização e investigação de marcadores genéticos para características de resistência poderiam acelerar o melhoramento genético e levar ao melhor entendimento dos mecanismos moleculares de resistência. O objetivo desse estudo foi verificar a possibilidade de seleção para características de resistência e resiliência em cordeiros da raça Morada Nova, estimar as potenciais respostas correlacionadas e avaliar se cinco polimorfismos de base única (SNPs, OAR2_14765360, OAR6_81718546, OAR11_62887032, OAR12_69606944 and OAR15_59871543) estão associados às características de resistência e resiliência. Um total de 287 cordeiros e 131 matrizes foram submetidas a dois desafios parasitários consecutivos e independentes por infecção oral com 4.000 larvas infectantes (L3) de Haemonchus contortus. Contagem de ovos por grama de fezes (OPG), volume globular (VG) e peso corporal vivo (PV) foram monitorados a cada uma ou duas semanas para 42 dias em cada desafio. Amostras de DNA de 287 cordeiros, 131 matrizes e 4 reprodutores machos foram amplificados por ARMS-PCR ou PCR-RFLP e os genótipos foram determinados. Estimativas de parâmetros genéticos foram obtidas para dias individuais de coleta, e para a característica como um todo com medidas repetidas, utilizando modelos animais mistos. A análise de variância (ANOVA) foi utilizada para análises de associação entre genótipos dos SNPs e fenótipos. No caso de associação significativa, o efeito de substituição de alelo foi calculado baseado em um modelo linear. As estimativas de herdabilidade para OPG no primeiro e segundo desafio parasitário foram, respectivamente, 0,25 ± 0,18 e 0,46 ± 0,19 para cordeiros, e 0,00 ± 0,09 e 0,20 ± 0,16 para matrizes. Para VG, as estimativas de herdabilidade foram 0,23 ± 0,14 e 0,32 ± 0,16 para cordeiros e 0.13 ± 0.11 e 0.37 ± 0.18 para matrizes. A estimativa de herdabilidade do ganho de peso diário (GPD) total foi 0,70 ± 0,21. As correlações genéticas de OPG e VG entre cordeiros e matrizes foram 0,36 ± 0,08 e 0,42 ± 0,08, respectivamente. Não foi encontrada correlação genética significativa entre GPD e as demais características, enquanto ocorreu uma correlação genética negativa entre OPG e VG (-0.70 ± 0.03). OAR2_14765360 e OAR12_69606944 foram associados com OPG, e OAR12_69606944 também teve um efeito significativo no VG e GPD, mostrando associação favorável do genótipo CC com todas as características avaliadas. OAR6_81718546 e OAR11_62887032 foram associados ao GPD, e OAR6_81718546 teve um efeito adicional no VG. OAR15_59871543 não foi polimórfico na população. OAR6_81718546 e OAR12_69606944 apresentaram efeito de substituição de alelo significativo de -1.06 ± 0.52 kg para o alelo T no PV final e 0.74 ± 0.32 para o alelo C no VG do mesmo dia de desafio, respectivamente. A seleção para OPG baixo e VG alto é possível em cordeiros da raça Morada Nova. A seleção para OPG baixo deve ter uma resposta correlacionada no VG (levando a VG mais alto), enquanto não é esperada uma resposta correlacionada no GPD. A seleção para OPG baixo e VG alto em cordeiros deve levar a OPG baixo e VG alto em matrizes. Um índice contendo GPD total e dados de duas coletas de OPG e VG (conduzidas 4 a 6 semanas após a infecção durante o segundo desafio parasitário) pode ser utilizado como critério de seleção em cordeiros, permitindo seleção simultânea para OPG mais baixo, VG mais elevado e GPD mais elevado. Nossos resultados suportam a existência de locos de características quantitativas (QTL) para resistência e resiliência em desequilíbrio de ligação com os SNPs polimórficos e sugere o seu uso futuro para a exploração dessas características em ovinos Morada Nova. Palavras-chave: marcadores genéticos, parâmetros genéticos, resistência do hospedeiro, resiliência, desafio parasitário 1 CHAPTER 1 – PRELIMINARY CONSIDERATIONS 1.1. INTRODUCTION Gastrointestinal nematodes (GIN) are a major constraint in sheep production worldwide (Perry et al. 2002). These parasites cause high economic losses due to their direct detrimental effects on health and performance of animals, and due to the necessity of costly treatments. This is especially true for tropical environments, which favour the survival and development of free stage larvae of some gastrointestinal nematodes, including Haemonchus contortus, the most important GIN in Brazilian sheep flocks. Its high pathogenicity is mainly due to hematophagia of the adult worms (and fifth stage larvae), which can lead to severe anaemia and subsequent deaths of infected animals. Many flock holders are highly dependent on the use of anthelmintic drugs to maintain their productivity (Jackson et al., 2009). The efficiency of this approach, however, is limited due to an increasing prevalence of drug-resistant parasites (Veríssimo et al., 2012; Papadopoulos et al., 2012; Albuquerque et al., 2017). In São Paulo state of Brazil, there are reports of flock holders abandoning sheep production due to this problem (Amarante et al., 2004). Several alternative strategies have been suggested in order to reduce the necessity of anthelmintic treatments and thus attenuate the development of anthelmintic resistance in parasites. Adequate nutrition and dietary supplementation, grazing management, targeted selective treatment (TST), biological control and the use of animals resistant or resilient to nematode infection are some examples, with the latter being one of the most promising approaches. The existence of variability in the ability of hosts to withstand GIN infections has been evidenced within and between sheep breeds in a number of studies (Baker and Gray, 2004; Aguerre et al., 2018; Snyman and Fisher, 2019). The feasibility and efficiency of genetic improvement of resistance is well-documented (Bisset et al., 2001; Eady et al., 2003; Kemper et al., 2010), and this trait has been included in commercial breeding programmes in Australia (Brown and Fogarty, 2017), New Zealand (Bisset et al., 2001) and Uruguay (Goldberg et al., 2012). Genetic parameters, especially correlations between resistance and performance traits, tend to vary across breeds and environments (Bishop and Stear, 2003), making it necessary to explore them separately for each situation. To obtain estimates of individual breeding values, it is also necessary to submit animals to either natural or artificial nematode infection, which can compromise performance and raise issues of comparability between environments and 2 generations (Hunt et al., 2008). Genetic markers for resistance and resilience could allow animals to be selected (in part or entirely) based on their genotypes. This could facilitate the selection process by reducing the need for phenotypic information and enhancing genetic prediction (Hunt et al., 2008; Rosa, 2011; Benavides et al., 2016). Morada Nova is a naturalized Brazilian hair sheep breed that is very well adapted to tropical conditions and has shown to be highly resistant and resilient to GIN infections (Facó et al., 2008; Issakowicz et al., 2016; Toscano et al., 2019). Furthermore, the Morada Nova’s aptitude as a maternal breed in meat production has been evidenced by Issakowicz et al. (2016), making this breed an important resource for efficient and sustainable production in tropical, highly parasitised environments. The aim of the present study is the genetic characterization of resistance and resilience to H. contortus in a Morada Nova population with the purpose of evaluating the feasibility of genetic selection and investigating underlying biological mechanisms. Genetic parameters for faecal egg counts (FEC), packed cell volume (PCV) and body weight, including phenotypic and genetic correlations between these traits, were estimated. In addition, five single nucleotide polymorphisms, formerly associated to FEC in a Red Maasai and Dorper packcross population (Benavides et al., 2015) were evaluated as for their association to FEC, PCV and body weight. 1.2. LITERATURE REVIEW 1.2.1. Relevance of gastrointestinal nematodes Gastrointestinal parasitism was estimated to be the most important animal health constraint based on a worldwide ranking elaborated by Perry et al. (2002), considering their wide geographical distribution, wide range of host species and their high economic impact on farms. The economic damage due to internal parasites was estimated to be 436 million $ AUD per year in the Australian sheep industry (Brown and Fogarty, 2017), with the greater proportion probably being caused by H. contortus (Emery et al., 2016). H. contortus is the most prevalent GIN in small ruminant flocks of Brazil (Chagas et al., 2013), including the state of São Paulo (Amarante et al., 2004). Apart from anaemia (which is due to the hematophagia of H. contortus), clinical signs of haemonchosis are oedemas (typically submandibular) caused by loss of plasmatic protein, anorexia, or, in severe cases, hemorrhagic gastritis. Even in cases of subclinical infections, performance is often constrained in terms of retarded growth, weight loss or low carcass quality. 3 H. contortus possesses a direct life cycle. The hosts are infected at grazing, by third stage larvae (L3) present on pastures. After shedding of their cuticle in the rumen, L3 larvae settle in apposition to the gastric glands of the abomasum, where they undergo the transformation to the fourth (L4) and fifth (L5) larval stage. L5 larvae develop an oral lancet that enables them to obtain blood from the mucosal vessels, briefly before their final transformation into adults. Eggs are eliminated with the faeces onto the pastures where the first stage (L1) larvae hatch within a few hours. Under favourable conditions, L3 may develop in 5 days, allowing the conclusion of a full life cycle within 20 days, the shortest of all GIN (Emery et al., 2016). This fact, together with an establishment rate of 60% and a daily egg output of 5000 to 15,000, guarantees H. contortus’s high ability of genetic adaptation, including acquisition of resistance to anthelmintic drugs (Emery et al., 2016). Counting of worm eggs in the animal’s faeces – faecal egg count (FEC) – is the measure most commonly used to access infection levels. It is a good indicator of worm burden of H. contortus (unless the worm burden is very high, which decreases fecundity) (Bishop et al., 2012b). FEC can range from zero to several thousand egg counts per gram of faeces. Within flocks, the majority of the animals have relatively low FEC, while a few animals shed a much higher number of eggs (Amarante et al., 1998). Therefore, the distribution of FEC is almost inevitably right-skewed, which is why most studies use some kind of transformation before performing analyses on FEC (Bishop et al., 2012b). Due to the hematophagia of H. contortus, packed cell volume (PCV) can be used to access the effect of infection on the health status of individual animals. PCV is a blood parameter that needs to be maintained within its physiological scope. With rising levels of H. contortus-infection, an increasing amount of blood is removed by the parasites, eventually causing anaemia. A negative correlation between FEC and PCV has consequently been related in a number of studies (Albers et al., 1987; Baker et al., 2003; Lôbo et al., 2009). The efficiency of anthelmintic treatments as a control measure for GIN is threatened by the increasing prevalence of drug resistant parasites, with multi-drug resistance against different classes of anthelmintics being frequently reported (Jackson et al., 2009; Zvinorova et al., 2016). Field studies have even revealed resistance of GIN to Monepantel, the most recent active component launched in 2009 (Scott et al., 2013; Mederos et al., 2014; Sales and Love, 2016). In São Paulo state, reduced efficiency of albendazole and ivermectin were found in 100%, of moxidectin in 96.6%, of closantel in 92.9% and of levamisole in 53.6% of the sheep flocks investigated by Veríssimo et al. (2012). As a result of this situation, mortality rates can rise despite anthelmintic treatments, causing the abandonment of sheep production (Amarante et al., 2004). The 4 implementation of alternative strategies for nematode control could reestablish the productivity of these production systems and attenuate the development of drug resistance in parasites. 1.2.2. Characteristics and potential of the Morada Nova breed Morada Nova is a naturalized hair sheep breed from the Northeast of Brazil that is very well adapted to the hot, semi-arid climate of this region. The first description of Morada Nova sheep was by Octávio Domingues, who first encountered sheep of this breed in 1937, in the municipality “Morada Nova” (Ceará), the supposed geographical centre of origin (Arandas, 2017). In the Northeast, Morada Nova sheep are an important resource for traditional, low-input production of meat and skin (Arandas et al., 2017). There are no records of the genetic origin of the Morada Nova breed, but contribution of both Mediterranean and African sheep have been suggested (Facó et al., 2008). Results of two studies using genome-wide genotyping of single nucleotide polimorphism (SNP) revealed a high genetic relatedness between Morada Nova and African breeds (Kijas et al., 2012; Toledo, 2014). Until 2006, the flock holder’s preference for larger sheep and indiscriminate crossbreeding with exotic breeds had caused a continuous decrease in the number of Morada Nova sheep. Since then, programmes with the aim of preservation, characterization and development of this breed have been implemented (McManus et al., 2019). In 2010, about 800 animals were registered by ARCO (Brazilian Association of sheep breeders), compared to about 400 animals in 2004. The Northeastern region of Brazil possesses the highest amount of Morada Nova sheep, but numbers are growing in the state of São Paulo. Between 350 and 400 of the 800 animals registered in 2010 were from Ceará, while both São Paulo and Paraiba accounted for 100 to 150 animals. Since 2012, São Paulo has surpassed the Northeastern states in numbers of registered animals, although the overall numbers have dropped considerably from 2010 to 2014, the last year of registration considered in this study (McManus et al., 2019). Due to the authors, this decline in numbers of registered animals is possibly related to costs for registration and a long drought in the Northeast, that began in 2011. Arandas et al. (2012) investigated the population structure of 36 Morada Nova herds, located in Ceará, Pernambuco, Paraíba, Rio Grande do Norte and São Paulo. Estimates of the effective population size (Ne), inbreeding rate (ΔF) and panmixia value (PAM) were calculated for each herd, obtaining a mean Ne of 12.88, ΔF of 0.08 and PAM 5 of 0.92, considering all flocks investigated. For the herds in São Paulo, means of 6.66 (Ne), 0.092 (ΔF) and 0.908 (PAM) were estimated. The authors concluded that the variability within the flocks of Morada Nova sheep was in danger, and suggest the implementation of a management plan for its maintenance. The relatively high heterozigosity, indicated by the panmixia value, might be a result of crossbreeding, rather than a within-breed phenomenon, due to the authors (Arandas et al., 2012). McManus et al. (2019) performed a pedigree analysis on 10,015 registered Morada Nova sheep, born between 1973 and 2014. Effective herd sizes ranged from 10 to 30 between 1983 and 2004. From 2004 to 2009, there was an increase in effective herd size, with a peak of over 70 N e for animals born between 2006 and 2009, and a subsequent slight decrease to a value of about 60 when considering animals born between 2008 and 2011. Genetic variability was found to be availble within the breed, given that the ratio between the effective number of ancestors (fa) and the effective number of founders (fe) was approximately 1. In the Santa Inês breed, this ratio was found to be 1.35, indicating a reduction in variability. The authors concluded that the genetic diversity of the Morada Nova breed is not immediately endangered (McManus et al., 2019). This breed’s characteristics are small size, high adaptation to tropical climate, high litter size, non-existent reproductive seasonality, good maternal ability and excellent pelt quality, but also low weight gain and carcass quality (Facó et al., 2008; Lôbo et al., 2011). The Morada Nova’s low adult body weight is considered advantageous by several authors (Facó et al., 2008; Lôbo et al., 2011; Issakowicz et al., 2016; McManus et al., 2019), because it results in higher stocking rates per hectare and higher production of kg lamb per kg feed. The latter is highly relevant insofar that feeding of the ewes constitutes the most important expense factor in lamb production (Lôbo et al., 2011). Economic values were estimated for a Morada Nova flock situated in a semi-arid environment of the Northeastern region of Brazil. This study showed that meat production was profitable under these circumstances and that improvement in carcass quality, slaughter weight, survival and some reproductive traits (like litter size) could increase the lucrativeness of the system. No significant economic value was found for the number of anthelmintic treatments in this study. A possible reason for this could be low parasite challenge (the very short rainy seasons during the experiment did not favour GIN development on pastures) or the natural resistance of Morada Nova sheep (Lôbo et al., 2011). In a study conducted by Issakowicz et al. (2016), ewes of the Santa Inês and Morada Nova breed showed low GIN infection level under natural parasite challenge as well as a high level of resilience. Even during the periparturient period – a phase in which 6 FEC usually increases due to a decrease in immunity (Kahn, 2003) – the ewe’s health was not affected. The Morada Nova ewes had lower FEC than Santa Inês ewes at 30 days post partum (mean values of 1114 and 2005, respectively). Also, they presented significantly higher PCV at 56 days of gestation as well as 30 and 60 days post partum (30.6 ± 1.1, 32.5 ± 1.5 and 33.5 ± 1.3%, respectively, for Morada Nova and 28.0 ± 1.1, 28.8 ± 1.5 and 30.9 ± 1.3% for Santa Inês ewes). Both breeds showed similarly high reproductive performance, with 1.5 lambs per ewe, a conception rate of 91 and 93 % and multiple births of 57 and 53% for Morada Nova and Santa Inês ewes, respectively. In addition, Morada Nova and Santa Inês ewes were mated either to rams of the same breed or to Dorper rams and measures of productivity were calculated for the pure-breed and cross-breed lambs. Santa Inês ewes are heavier than Morada Nova ewes (51.8 ± 7.07 kg and 33.1 ± 4.98, respectively), and they produced lambs with higher body weight at birth (6.20 ± 0.4 kg and 4.27 ± 0.8 kg, respectively) and at weaning (26.9 ± 2.0 kg and 18.9 ± 1.9 kg, respectively). However, when crossed with Dorper rams, the Morada Nova ewes produced significantly higher amounts of litter weight at weaning per kg body weight of dam (0.62 ± 0.03 kg) compared to Santa Inês X Dorper (0.47 ± 0.03 kg), Santa Inês X Santa Inês (0.42 ± 0.03 kg) and Morada Nova X Morada Nova (0.43 ± 0.03 kg), showing its potential as a maternal breed in meat production (Issakowicz et al., 2016). In a study that compared Strongylida FEC of several breeds and crosses under natural infection, animals of the Morada Nova breed had the lowest FEC (mean FEC of 8.65), compared to Bergamasca (193.99), Santa Inês (347.88), Ile de France (484.62), Ile de France X Santa Inês (482.26) and Texel X Santa Inês (279.50) (McManus et al., 2009). These results are consistent with the observations made in the Morada Nova flock at the Embrapa Southeast Livestock Unit (CPPSE) in Brazil, that showed these animals to be extremely resistant to GIN, evidenced by a low number of anthelmintic treatments required. In monthly samplings, Morada Nova sheep consistently showed higher PCV than animals of the other breeds kept under equal management conditions (36.3% for Morada Nova, 29.5% Santa Inês, 25.9% Dorper and 26.9% Texel) (Chagas et al., 2015). Resistance and resilience to nematodes are abilities of great interest to sheep production systems in Brazil, given the economic relevance of GIN in the tropics and the problem of drug resistant parasites. The Morada Nova breed could be a tool to increase efficiency and sustainability of sheep production. 1.2.3. The host’s response to parasites Resistance is defined by Bishop and Stear (2003) as the “ability of the host to 7 resist infection or to control the parasite life cycle”. Measures to quantify resistance can be worm burden, worm size, fecundity and FEC (Bishop, 2012b), with the latter being the most commonly used. Apart from resistance, other aspects of the host’s response to parasites are of interest. Especially for parasites with high prevalence (where a high percentage of the flock is infected) the performance of a flock will depend on the ability of the individuals to minimize the damage that the infection causes on their health and performance status. Tolerance is defined as the “net impact on performance of a given level of infection” (Bishop 2012b). In other words: an animal that is resistant to parasites is able to decrease the level of infection, while a tolerant animal is able to decrease the detrimental effect of infection on its performance. Tolerance is difficult to estimate on the individual animal level and for this purpose, the infection status (measured by FEC) has to be taken into account. Breeding values for tolerance can be estimated when related animals graze pastures of different (and well-defined) contamination levels (Bishop, 2012b), although no empirical example of this was found for tolerance of nematodes in sheep. Resilience, the productivity of an animal in the face of infection (Bishop,2012b), is closely related to tolerance and both terms are used synonymously by some authors. Bishop and Stear (2003) stated that resilience, often measured as a performance trait under parasite challenge conditions, is a combination of resistance, tolerance and performance. Tolerance could thus be considered resilience in the narrow sense. Examples for performance traits quantified under parasite challenge as a measure of resilience are body weight or weight gain (Albers et al., 1987), but the necessity of anthelmintic treatments (Bisset et al., 2001; Morris et al., 2010) or indicators of anaemia in the case of H. contortus (Baker et al., 2003; Aguerre et al., 2018; Oliveira et al., 2018) have also been treated as resilience traits. Resistance, resilience and tolerance are results of defense mechanisms – primarily the immune response – but also of hemostasis, hematopoesis and other mechanisms linked to maintenance and recovery of the integrity of tissues. These mechanisms require resources and energy, and they are highly dependent on the environment (particularly nutrition) and physiological status of the animal (Bishop and Stear, 2003). Ewes usually undergo a considerable increase in GIN infection during the periparturient period (Miller et al. 1998; Matika et al., 2003; Williams et al., 2010), turning it the most significant phase in pasture contamination (Williams et al., 2010). This effect is caused by a temporary decrease in immunity, which is probably due to an increased requirement for energy and nutrients (essentially protein), with the partial priority of the gut immune system for protein supply being reduced in favour of the maintenance of 8 pregnancy and lactation (Kahn, 2003). GIN infections decrease feed intake, efficiency of feed use and protein levels, with protein being lost into the gastrointestinal tract and increased requirement of protein for repair of affected tissues, maintenance of plasma protein levels and mucoprotein production (Coop and Holmes, 1996). Thus, protein supplementation attenuates the effects of GIN infection and favours an effective immune response (Coop and Holmes, 1996; Kahn, 2003). Regarding susceptibility to GIN infections, an important factor is age, with lambs consistently showing higher infection levels than adults of the same breed (Woolaston and Piper, 1996; Vanimisetti et al., 2004; Goldberg et al., 2012). Apart from the fact that younger animals have less previous contact with parasites and thus had less time to mount an effective immune response against them, immunological hyporesponsiveness in young animals has also been discussed (Colditz et al., 1996). Another factor with great importance for susceptibility to GIN is sex: male animals are frequently reported to have higher FEC in the face of GIN infection, and lower PCV in the case of H. contortus (Eady et al., 2003; Gauly et al., 2006; Snyman and Fisher, 2019), and a positive correlation between testosterone levels and worm burden was related by Gauly et al. (2006). 1.2.4. Epidemiological factors of resistance to GIN Within sheep flocks, highly susceptible animals comprise a large amount of parasites, being responsible for a disproportionately high fraction of larval pasture contamination (Amarante et al., 1998). Resistant animals, on the contrary, eliminate less parasite eggs, which leads to low levels of pasture contamination and decreases the infection pressure within the flock (Bishop and Stear, 2003). This effect was first evidenced in Romney lambs by Bisset et al. (1997), who showed that on pastures grazed by animals of a high-FEC selection line (susceptible), contamination with Trichostrongylus larvae was 5 to 6-fold compared to pastures grazed by animals selected for low FEC (resistant), leading to higher weight gains in the resistant group. Morris et al. (2005) observed that a superior performance of susceptible animals in weight gain and wool traits compared to resistant animals was levelled when these two groups were managed in different pastures. Williams et al., (2010) found similar results in periparturient Australian Merino ewes, showing lower infection levels and reduction of pasture contamination with T. colubriformis larvae in a group selected for resistance compared to an unselected control group. Consequently, lambs of resistant ewes have the advantage of an environment with low contamination levels, promoting performance (Williams et al., 2010). In a mathematical model implemented by Bishop and Stear (1999), increase in 9 weight gain after selection for low FEC was twice as high as would have been expected based on additive genetic effects, suggesting that a considerable part of the increase was due to epidemiological benefits (reduced contamination of pastures by resistant animals). Using a similar model, Laurenson et al. (2012) estimated that susceptible lambs can benefit from contemporary grazing with resistant animals, reaching higher body weights (BW) than when grazed separately, while the resistant lamb’s performance is not impaired. 1.2.5. Basic concepts of animal breeding Animal breeding has the aim of improving the performance of future generations, which is achieved by selecting and mating animals that are considered superior in one or several traits, in comparison to the average population. One precondition for selection is the existence of variation: animals have to differ in their performance, or else it would not be possible to select the “best” animals. If individuals differ in their body weight, for example, it is possible to select and mate only the heaviest animals. However, to achieve genetic merit, the respective trait needs to be – at least to some extent – heritable. Essentially, heritability is the correlation between phenotypic and genetic values of a trait. If the heritability is high, the phenotype of an animal is a good indicator of its genetic merit. In this case, animals of high phenotypic values for a trait tend to have progeny that also (in average) present high phenotypic values for this trait. Heritability estimates can take values between 0 and 1. Traits with heritability estimates of 0 to 0.2 are considered lowly, those of 0.2 to 0.4 moderately, and above 0.4 highly heritable (Bourdon, 2000). 1.2.6. Selection of animals resistant to GIN infection Because of its easy application, FEC is the measure most commonly used for GIN resistance quantification in sheep. For animals to express their genetic potential for resistance, a certain level of infection is indispensable. Therefore, animals have to be submitted to either natural or artificial infection in order to permit genetic selection (at least when selection is based on phenotypes). The existence of genetic variation in FEC has been evidenced by many studies, both after natural (Gowane et al., 2019 Brown and Fogarty, 2017; Snyman and Fisher, 2019) and artificial (Woolaston and Piper, 1996; Morris et al., 2005; Aguerre et al., 2018) parasite challenge and in a number of sheep breeds, including Australian Merino (Brown and Fogarty, 2017), Perendale (Morris et al., 2005), Dohne Merino (Snyman and Fisher, 2019), Avakalin and Malpura sheep (Gowane et al., 10 2019), Blond-faced Manech (Aguerre et al., 2018), Scottish Blackface (Bishop et al., 1996), Texel (Bishop et al., 2004) and Santa Inês (Oliveira et al., 2018). Most studies related moderate heritability of FEC, although estimates differed considerably between genetic groups and environments. The feasibility of selection for FEC is well-documented. Lines of resistant animals were successfully implemented by means of selection based on artificial challenge with H. contortus in Merino (Woolaston and Piper,1996) and based on natural mixed infection in Perendale (Morris et al., 2005) and Rylington Merino (Karlsson and Greeff, 2006) sheep. Moderate heritabilities and high phenotypic variation of FEC permit noticeable genetic progress of this trait (Eady et al., 2003; Kemper et al., 2010; Brown and Fogarty, 2017). For the Rylington Merino selection line, Kemper et al. (2010) found that FEC of resistant sheep was approximately 82% lower compared to unselected animals, after 15 years of selection. Eady et al. (2003) detected a 69% reduction of FEC in animals of the selected Merino line described by Woolaston and Piper (1996), compared to an unselected group. These authors also related that FEC was lower in animals that were not drenched, but selected for resistance than in unselected animals under a regular drenching regime (Eady et al., 2003). Selection is often based on FEC records in lambs (Woolaston and Piper,1996; Morris et al., 2005; Karlsson and Greeff, 2006). However, given that the periparturient period is the most significant phase in larval pasture contamination (due to the temporary decrease in ewe’s immunity described earlier), higher resistance in periparturient ewes is a desirable goal, leading to reduced larval challenge for lambs (Williams et al., 2010). The genetic correlation between FEC in lambs and FEC in periparturient ewes under natural infection was estimated to be 0.81 ± 0.11 in Uruguayan Merino sheep (Goldberg et al., 2012) and Williams et al. (2010) related that animals from the Rylington Merino selection line (Karlsson and Greeff, 2006), selected for low FEC as lambs, remained low infection levels later in life, during the periparturient period. These authors concluded that, in the respective sheep populations they investigated, FEC in lambs can be used as a criterion to select for more resistant ewes, avoiding sampling during the periparturient period, which is often stressful for the animals. On the other hand, Vanimisetti et al. (2004) did not detect a significant genetic correlation between FEC in lambs and FEC in ewes in a Dorset X Rambouillet X Finnsheep population after artificial infection with H. contortus. Another approach to indirectly access resistance in ewes was described by Aguerre et al. (2018): instead of selecting lambs, these authors suggested the selection of rams after artificial challenge with H. contortus. The genetic correlation of 0.56 ± 0.01 to 0.71 ± 0.01 between FEC in artificially infected rams and FEC in ewes under natural infection on 11 pasture confirmed that selection for low FEC in rams (which is more easily implemented in this production system than selection of ewes) should, in the future, increase resistance of ewes (Aguerre et al., 2018). Another issue is the question of whether or not resistance to different GIN species is correlated. In a study conducted on Texel lambs, Bishop et al. (2004) found that the genetic correlation between Nematodirus FEC and Strongyle FEC (including the genera Oesophagostomum, Chabertia, Bunostomum, Trichostrongylus, Cooperia, Ostertagia, Teladorsagia and Haemonchus) ranged between 0.38 and 0.95 on different sampling dates. Gruner et al. (2004) compared resistance to Trichostrongylus colubriformis and H. contortus by artificially infecting two groups of animals with both parasite species in subsequent challenges. One group was first infected with H. contortus, then drenched, and subsequently infected with T. colubriformis. The other group was also infected with both species, but in reverse order. Genetic correlations between the two groups for FEC of different species were predominantly close to 1. These results suggest that resistance against GIN is not species-specific. Selection for resistance to one species should therefore lead to genetic progress in resistance to other GIN species (Gruner et al., 2004). 1.2.7. Selection of animals resilient to GIN infection The level of anaemia that an animal shows when submitted to hematophagous H. contortus depends on its resistance: highly resistant animals have lower FEC and worm burdens, with less blood being removed by parasites. On the other hand, some animals are able to maintain blood parameters within the physiological scope even when heavily parasitized, which is why most authors consider measures of anaemia to be resilience traits (Baker et al., 2003; Bishop, 2012; Aguerre et al., 2018). The existence of genetic variance of PCV after H. contortus infection in sheep has been evidenced in several studies (Baker et al., 2003; Lôbo et al., 2009; Oliveira et al., 2018). Baker et al. (2003) estimated the heritability of PCV to be 0.14 ± 0.05 in a population consisting of 6 – 8 month old Red Maasai, Dorper and Red Maasai X Dorper lambs after natural mixed infection, with H. contortus being predominant (73%). A heritability of 0.39 was estimated for PCV in lambs and 0.15 in ewes after a single artificial infection with H. contortus (Vanimisetti et al., 2004). For the Santa Inês breed, Oliveira et al. (2018) found a heritability estimate of 0.30 ± 0.06 under natural challenge, while Lôbo et al. (2009) conducted two subsequent artificial infections with H. contortus in lambs of the same breed, with a heritability peak of 0.31 in the first and 0.12 in the second parasite challenge. 12 A widely-used measure for anaemia in small ruminants is the Famacha© score, which allows the assessment of the degree of anaemia by comparison of the colour of the animal’s eye mucosa to the Famacha© chart. This method has been successfully implemented as a criterion for targeted selective treatment (TST) in a number of tropical sheep farming systems (Burke et al., 2007), although there are studies that claim it is not suitable for some breeds (Moors et al., 2009; Bishop 2012a). Also, Cintra et al. (2018) highlighted that the sensitivity of this method was low in lambs (30.8 % when Famacha© 3-5 and PCV of < 18% were considered anaemic). Heritability estimates for the Famacha© score were 0,29 ± 0.05 in a Dohne Merino flock (Snyman and Fisher, 2019), and 0.21 ± 0.04 for Santa Inês lambs (Oliveira et al., 2018). Measures of anaemia, like PCV and Famacha©, consistently have favourable phenotypic correlations with FEC (that is, PCV is negatively and Famacha© positively correlated with FEC) (Baker et al., 2003; Lôbo et al., 2009; Snyman and Fisher, 2019). While Lôbo et al. (2009) and Aguerre et al., (2018) found no significant genetic correlation between FEC and PCV, other studies related genetic correlations that ranged from – 0.63 ± 0.58 to – 0.98 ± 0.24 (Albers et al. 1987; Baker et al., 2003). Genetic correlations of the Famacha© score with FEC were estimated to be favourable in Santa Inês (0.28 ± 0.03) (Oliveira et al., 2018), as well as in the Dohne Merino Flock (0.62 ± 0.08) (Snyman and Fisher, 2019). Body weight and weight gain in the face of GIN infection are appealing breeding goals, given that animals are continually exposed to parasites in many production systems (Bishop, 2012a; Bishop, 2012b). Albers et al. (1987) compared growth rates of both infected and uninfected animals, and related that heritability estimates for weight gain (WG) in infected animals and WG depression under infection were low, with high standard error (0.15 ± 0.08 and 0.09 ± 0.07, respectively). The same was true for genetic correlations between WG depression and resistance (0.36 ± 0.34 and 0.31 ± 0.36) (Albers et al., 1987). Another approach to assess resilience was described by Morris et al. (2010). These authors defined resilience as the age at which an anthelmintic treatment was necessary to maintain acceptable growth rates under natural parasite challenge. Animals were drenched based on their weight gain compared to a control group of uninfected animals. The heritability estimate of the described trait was 0.13 ± 0.02. After 13 years of selection, the age at first drench was increased by 23.6 days in animals of the resilience selection line compared to an unselected control group and resulted in a 4.5 kg increase in 6-month life weight. The genetic correlation between resilience and FEC (resistance) was not significantly different from zero. Consequently, the FEC in the resilience line did not differ from that of the control line, suggesting that resistance and resilience are based on 13 different biological mechanisms (Morris et al., 2010). Selection based on resilience traits is a controversial issue: If animals were selected solely based on resilience traits similar to that described by Morris et al. (2010), resistance would not be affected (Albers et al., 1987; Bisset et al., 2001). In this case, animals would shed the same amount of eggs and resilience mechanisms could break down in phases of high larval contamination levels, leading to high morbidity. On the other hand, resilience is a mechanism that does not interfere with the parasite life-cycle, and thus imposes low selective pressure on the parasite, lowering the risk of an eventual overcoming of the host’s response mechanisms. High resilience is frequently described in breeds that have adapted to a high challenge environment (Bishop and Stear, 2003). Baker et al. (2003) affirmed that in Red Maasai sheep, which are highly resistant, improvement of resilience (measured as PCV) should be given more weight than resistance, while in the Dorper breed, which is more susceptible, both traits should be selected for. However, the resilience trait that is cited here – PCV – has a moderate negative correlation with FEC (- 0.34), which would lead to a correlated response in resistance, even if it is not directly selected for (Baker et al., 2003). Baker and Gray (2004) also suggested to select the heaviest rams under parasite challenge or to use the Famacha© score as a culling criterion in Red Maasai sheep, as a simple breeding programme for small flock holders who do not have resources for FEC or PCV sampling. Bishop (2012a) considers selection indices which include resistance, performance under parasite challenge and measures of anaemia to be a sensible approach for sheep production in the tropics. In summary, the usefulness of selection towards higher resilience probably depends on the resilience trait in question, whether other traits are also being selected for, as well as on the breed and on environmental factors. 1.2.8. Genetic correlations between resistance and performance Because of the importance of estimating correlated responses prior to selection for resistance traits, the genetic relationship between FEC and weight or weight gain has been studied in a variety of environments and breeds. There has been little consistency across studies: negative genetic correlations were related by Bishop et al. (1996) (-0.63 – -1.00), Snyman and Fisher (2019) (-0.39 ± 0.22), Oliveira et al. (2018) (-0.27 ± 0.03) and Benavides et al. (2016) (-0.05 ± 0.00), while Lôbo et al. (2009) and Brown and Fogarty (2017) found no significant correlations, and positive correlations were detected by Morris et al. (1997) (0.05 ± 0.04 and 0.07 ± 0.05) and Gowane et al. (2019) (0.35 ± 0.16). A mathematical model, implemented by Bishop and Stear (1999), that simulated different 14 scenarios of host-parasite interaction in a sheep population, considering breed, diet, parasite species, pathogenicity and epidemiological factors, genetic correlations between FEC and growth traits increased from -0.02 to -0.46, simultaneously with an increase in disease severity. According to Bishop and Stear (2003) and Bishop (2012), the relationship between resistance and performance traits is highly dependent on the environment: in heavily parasitized environments, the effort of building and maintaining an effective immune response is beneficial as it leads to lower FEC and subsequent better performance of resistant animals. Under low challenge conditions, performance may not be compromised by infection, which makes the mounting of an immune response an unnecessary waste of energy and resources, leading to poorer performance of resistant animals. 1.2.9. Variation between breeds and genotype by environment interactions Variation of resistance and resilience has not only been found within populations, but it has frequently been reported between sheep breeds as well. (Baker and Gray, 2004). Resistant and resilient sheep breeds are typically native or naturalized breeds that have evolved in tropical environments, under high levels of parasite challenge, like Red Maasai (Baker et al., 2004), Sabi (Matika et al., 2003), Gulf Coast Native (Miller et al., 1998), Santa Inês (Amarante et al., 2004) and Morada Nova (Issakowicz et al., 2016). These breeds are usually characterized by low performance, which is probably due to low levels (or absence) of artificial selection and not necessarily to their lack of potential (Bishop and Stear, 2003). Flock holders in tropical countries often choose high performing commercial breeds. However, in some situations, these breeds do not express their potential for performance because they lack environmental adaptation. One example is the genotype by environment interaction documented by Baker et al. (2004) in Red Maasai and Dorper sheep: The Dorper breed, developed under semi-arid conditions in South Africa, performed equally well or better compared to the red Maasai breed when kept under semi-arid conditions. When both breeds were kept on a farm in a region of sub- humid climate, however, Red Maasai sheep showed superior performance. Apart from the higher resistance and resilience to GIN (evidenced by lower FEC and higher PCV values compared to the Dorper sheep), higher tolerance of heat and humidity, and the ability to utilize poor quality feed could also be of importance for the higher performance of Red Maasai sheep (Baker et al., 2004). 15 1.2.10. Genetic markers Quantitative trait loci (QTL) are chromosomic regions contributing to variation in phenotypic traits. Information on QTL can provide an understanding of molecular mechanisms that lead to variation in traits (Benavides et al., 2016; Ali et al., 2019) and it can be used for prediction of genetic merit (Hunt et al., 2008; Rosa 2013). Direct genetic markers are known polymorphisms in the DNA sequence within QTLs that have a direct causative effect on the trait under investigation (functional polymorphism). “LD-markers“, on the other hand, are known polymorphisms which are not themselves causative but are located in close proximity to the causative mutation and are therefore very likely to be simultaneously passed on to the next generation (Rosa, 2013). If two loci are likely to be inherited together, they are in so-called linkage disequilibrium – LD with each other, which means that a crossing over (the recombination of loci between homologous chromosomes during meiosis) between these loci is unlikely. Once implemented in a population, both marker types provide information on a trait: based on the genotype of an animal and at least a part of the phenotypic variation or genetic merit of this animal can be predicted. The utilization of genetic markers for selection (marker-assisted selection – MAS) is especially advantageous in traits of low heritability, that are difficult to measure, that are expressed late or only once in the animal’s life (like carcass quality) or that are expressed in only one sex (like milk yield) (Rosa, 2013). In simple-inherited traits or in the case of genes with a major effect on a trait, selecting individuals with a favourable marker genotype is straightforward. For litter size in sheep, a mutation in a major gene (Bone Morphogenetic Protein IB Receptor (BMPR1B)) was identified in highly prolific Boorola sheep (Wilson et al., 2001). Given the importance of this gene’s effect, selection for increased litter size can be based on a single marker information, as described by Chen et al. (2015). For most traits, however, MAS is more complex. If several combined markers account for a considerable proportion of phenotypic variance, information on animal’s genotypes for these markers can be used as an addition to breeding values (EBV) estimated in quantitative proceedings, either by including marker information into the calculation of EBVs, or by implementing a separate culling threshold, culling animals with undesirable genotypes and then applying selection on the remaining animals based on EBVs (Hunt et al., 2008). Another approach for marker-assisted selection is the utilization of a large number of markers spread throughout the genome (Genome-wide Marker-Assisted Selection (GWMAS), or Genomic Selection (GS)). For this purpose, it is common to use indirect markers provided by precast marker chips. Indirect markers are not necessarily in LD with 16 causative mutations and therefore, are not associated to the trait of interest. However, when marker distribution is sufficiently dense throughout the genome, the pedigree structure of the population can be derived from the marker information. This pedigree structure can then be integrated into EBV calculation in order to enhance prediction of genetic merit (Genomic Selection) (Rosa, 2013). There are different methods to detect LD or direct markers (although it is far less likely to detect a direct marker than an LD marker). Variants of genes that are known or supposed to be involved in biological processes associated to the respective trait can be tested for their aptitude as genetic markers (candidate gene approach; Schwaiger et al., 1995; Coltman et al., 2001; Ali et al., 2019). Genome wide association studies do not make any prior assumptions of relevant genes or regions, but perform a search of the whole genome, based on SNP markers, to identify regions with association to the respective trait (QTLs) (Kemper et al., 2011; Benavides et al., 2015; Berton et al., 2017). 1.2.11. Genetic markers for resistance to GIN in sheep Results on QTL for resistance to GIN in sheep are not always consistent throughout studies. According to Zvinorova et al. (2016), this is due to diverging methods of QTL detection as well as to the very variable circumstances under which the studies are conducted. Different parasite species have been investigated in various breeds and at different ages, using animals of only one or both sexes and of varying physiological status, which makes comparison of study results problematic (Zvinorova et al., 2016). Another issue is the reconstruction of causative genes in LD to significant markers, given that annotation of the sheep genome is still in progress (Benavides et al., 2016). The regions most consistently associated to nematode resistance are those containing the major histocompatibility complex II (MHC II) gene on chromosome 20 (Schwaiger et al., 1994; Janssen et al., 2002; Ali et al., 2019) and the interferon γ (IFNγ) gene on chromosome 3 (Coltman et al., 2001; Sayers et al., 2005). Both proteins are involved in the immune response, with MHC II being an antigen-presenting receptor and IFNγ being an inhibitor of the Th2 response, favouring the Th1 pathway (Coltman et al., 2001). The Th1 response was shown to be associated to H. contortus susceptibility in sheep (Zaros et al., 2010). In a review by Benavides et al. (2016), the Th2 response has also been suggested as relevant for GIN resistance, given that regions containing genes of the Th2 response were associated to FEC in several studies, including genes of the eosinophilia, mastocytosis and immunoglobulin E (IgE) pathways. Abomasal mucus 17 production is a part of the innate immune response against gastrointestinal parasites and genetic markers with proximity to genes involved in the glykosylation of mucins (the main component of mucus) have been associated to parasite resistance in several studies (Benavides et al., 2016). A third mechanism that is suggested to be important by these authors is hemostasis, with regions containing genes of hemostasis pathways being frequently reported as relevant. In a GWAS performed by Berton et al. (2017) on Santa Inês sheep, regions associated to FEC contained genes of immune response pathways, including the IL15, a known activator of the Th2 response (Toscano, 2019). Results of the same study also pointed to iron transportation and construction pathways (Berton et al., 2017). Benavides et al. (2015) conducted a GWAS study on animals of extreme phenotypes for resistance, selected from a Red Maasai and Dorper backcross population. Associations with FEC, PCV and body weight under parasite challenge were evaluated. The five most relevant SNPs associated to FEC, located on the chromosomes 2, 6, 11, 12 and 15 (OAR2_14765360, OAR6_81718546, OAR11_62887032, OAR12_69606944 e OAR15_59871543), were responsible for 2,33 % of phenotypic variation. None of these SNPs was associated to PCV or body weight. The following two chapters will present the experimental design, statistical methods, results and discussion of genetic parameters (chapter 2) and association analyses of five SNPs (chapter 3) for FEC, PCV and weight gain in Morada Nova sheep under H. contortus infection. 1.3. REFERENCES Aguerre S, Jacquiet P, Brodier H, Bournazel JP, Grisez C, Prévot F, Michot L, Fidelle F, Astruc JM, Moreno CR (2018) Resistance to gastrointestinal nematodes in dairy sheep: Genetic variability and relevance of artificial infection of nucleus rams to select for resistant ewes on farms. Veterinary Parasitology 256:16-23. Albers GAA, Gray GD, Piper LR, Barker JSF, Le Jambre LF, Barger IA (1987) The genetics of resistance and resilience to Haemonchus contortus infection in young Merino sheep. International Journal for Parasitology 17:1355-1363. Albuquerque ACA, Bassetto CC, Almeida FA, Amarante AFT (2017) Development of Haemonchus contortus resistance in sheep under suppressive or targeted selective treatment with monepantel. Veterinary Parasitology 246:112-117. Ali AOA, Murphy L, Stear A, Fairlie-Clarke K, Brujeni GN, Donskow Łysoniewska K, Groth‐ D, Buitkamp J, Stear MJ (2019) Association of MHC class II haplotypes with reduced faecal nematode egg count and IgA activity in British Texel sheep. Parasite Immunology https://doi.org/10.1111/pim.12626 https://doi.org/10.1111/pim.12626 18 Amarante AFT, Godoy WAC, Barbosa MA (1998) Nematode egg counts, packed cell volume and body weight as parameters to identify sheep resistant and susceptible to infections by gastrointestinal nematodes. Ars Veterinaria 14:331-339. Amarante AFT, Bricarello PA, Rocha RA, Gennari SM (2004) Resistance of Santa Inês, Suffolk and Ile de France sheep to naturally acquired gastrointestinal nematode infections. Veterinary Parasitology 120:91-106. Arandas JKG (2017) Etnozootecnia da raça ovina Morada Nova em seu centro de origem: história, critérios de seleção e sistema de produção. 137 pp. Thesis (PhD of Animal Science) – Federal Rural University of Pernambuco Arandas JKG, Ribeiro MN, Pimenta Filho EC, da Silva RCB, Facó O, Esteves SN (2012) Estrutura populacional de ovinos da raça Morada Nova. In: SIMPÓSIO BRASILEIRO DE MELHORAMENTO ANIMAL, 9., 2012, João Pessoa. Anais... João Pessoa: SBMA, 2012. 3 f. 1 CD-ROM. Baker RL, Nagda S, Rodriguez-Zas SL, Southey BR, Audho JO, Aduda EO, Thorpe W (2003) Resistance and resilience to gastro-intestinal nematode parasites and relationships with productivity of Red Maasai, Dorper and Red Maasai ✕ Dorper crossbred lambs in the sub-humid tropics. Animal Science 76:119-136. Baker RL, Gray GD (2004) Appropriate breeds and breeding schemes for sheep and goats in the tropics. In,: Sani RA, Gray GD, Baker RL (Eds.) Worm Control for Small Ruminants in Tropical Asia. Canberra, Australia: ACIAR Monograph 113, pp. 63-95. Baker RL, Mugambi JM, Audho JO, Carles AB, Thorpe W (2004) Genotype by environment interactions for productivity and resistance to gastro-intestinal nematode parasites in Red Maasai and Dorper sheep. Animal Science 79:343-353. Benavides MV, Sonstegard TS, Kemp S, Mugambi JM, Gibson JP, Baker RL, Hanotte O, Marshall K, Tassel C (2015) Identification of Novel Loci Associated with Gastrointestinal Parasite esistance in a Red Maasai x Dorper Backcross Population. PLOS one 10:1-20. Benavides MV, Sonstegard TS, Van Tassell C (2016) Genomic regions associated with sheep resistance to gastrointestinal nematodes. Trends in Parasitology 32:470–480. Berton MP, de Oliveira Silva RM, Peripolli E, Stafuzza NB, Martin JF, Álvarez MS, Gavinã BV, Toro MA, Banchero G, Oliveira PS, Eler, JP (2017) Genomic regions and pathways associated with gastrointestinal parasites resistance in Santa Inês breed adapted to tropical climate. Journal of Animal Science and Biotechnology 8:73. Bishop SC (2012a) Possibilities to breed for resistance to nematode parasite infections in small ruminants in tropical production systems. Animal 6:741-747. Bishop SC (2012b) A consideration of resistance and tolerance for ruminant nematode infections. Frontiers in Genetics 3:168/1-7. Bishop SC, Jackson F, Coop RL, Stear MJ (2004) Genetic parameters for resistance to nematode infections in Texel lambs and their utility in breeding programmes. Animal Science 78:185-194. 19 Bishop SC, Stear MJ (2003) Modeling of host genetics and resistance to infectious diseases: understanding and controlling nematode infections Veterinary Parasitology 115:147-166. Bishop SC, Morris CA (2007) Genetics of disease resistance in sheep and goats. Small Ruminant Research 70:48-59. Bisset SA, Vlassof A, West CJ, Morrison L (1997) Epidemiology of nematodosis in Romney lambs selectively bred for resistance or susceptibility to nematode infection. Veterinary Parasitology 70:255-269. Bisset SA, Morris CA, McEwan JC, Vlassof A (2001) Breeding sheep in New Zealand that are less reliant on anthelmintics to maintain health and productivity. New Zealand Veterinary Journal 49:236-246. Bourdon, R.M. (2000) Understanding animal breeding. Upper Saddle River, NJ: Prentice Hall. 418 pp Brown DJ, Fogarty NM (2017) Genetic relationships between internal parasite resistance and production traits in Merino sheep. Animal Production Science 57:209-215. Chagas ACS, Katiki, LM, Silva, IC, Giglioti R, Esteves SN, Oliveira MCS Barioni-Júnior W, (2013) Haemonchus contortus: A multiple-resistant Brazilian isolate and the costs for its characterization and maintenance for research use. Parasitology International 62:1–6. Chagas ACS, Zaia MG, Domingues LF, Rabelo MD, Politi FAZ, Anibal FF, Chagas JR (2015) Desparasitación racional: estudio comparativo de técnicas para la detección de la anemia causada por nemátodos gastrointestinales en pequeños rumiantes. In: VII Congreso Argentino de Parasitología, Libro de Resúmenes… La Plata: Associación Parasitológica Argentina, p. 109. Chen X, Sun H, Tian S, Xiang H, Zhou L, Dun W, Zhao X (2015) Increasing litter size in a sheep breed by marker-assisted selection of BMPR1B A746G mutation. Journal of genetics 94:139-142. Colditz IG, Watson DL, Gray GD, Eady SJ (1996) Some relationships between age, immune responsiveness and resistance to parasites in ruminants. International Journal for Parasitology 26:869-877. Coltman DW, Wilson K, Pilkington JG, Stear MJ, Pemberton JM (2001) A microsatellite polymorphism in the gamma interferon gene is associated with resistance to gastrointestinal nematodes in a naturally-parasitized population of Soay sheep. Veterinary Parasitology 122:571-582. Coop and Holmes (1996) Nutrition and Parasite Interaction. International Journal for Parasitology 26:951-962. Eady SJ, Woolaston RR, Barger IA (2003) Comparison of genetic and nongenetic strategies for control of gastrointestinal nematodes of sheep. Livestock Production Science 81:11-23. Emery DL, Hunt PW, Le Jambre LF (2016) Haemonchus contortus: the then and now, and where to from here?. International Journal for Parasitology. 46(12):755-69. 20 Facó O, Paiva SR, Alves LRN, Lôbo RNB, Villela LCV (2008) Raça Morada Nova: Origem, Características e Perspectivas. Embrapa Documentos 75 ISSN 1676-7659. Gauly M, Schackert M, Hoffmann B, Erhardt G (2006) Influence of sex on the resistance of sheep lambs to an experimental Haemonchus contortus infection. DTW. Deutsche tierärztliche Wochenschrift 2006 113:178-181. Goldberg V, Ciappesoni G, Aguilar I (2012) Genetic parameters for nematode resistance in periparturiant ewes and post-weaning lambs in Uruguayan Merino sheep. Livestock Science 147:181-187. Gowane GR, Swarnkar CP, Misra SS, Kumar R, Kumar A, Prince LLL (2019) Genetic parameter estimates for fecal egg counts and their relationship with growth in Avikalin and Malpura sheep. Animal 13:1788-1796. Gruner L, Bouix J, Brunel JC (2004) High genetic correlation between resistance to Haemonchus contortus and to Trichostrongylus colubriformis in INRA 401 sheep. Veterinary Parasitology 119:51-58. Hunt PW, McEwan JC, Miller JE (2008) Future perspectives for the implementation of genetic markers for parasite resistance in sheep. Tropical Biomedicine 25:18–33. Issakowicz J, Issakowicz ACKS, Bueno MS, Costa RLD, Katiki LM, Geraldo AT, Abdalla AL, McManus C, Louvandini H (2016) Parasitic infection, reproductive and productive performance from Santa Inês and Morada Nova ewes. Small Ruminant Research 136:96-103. Jackson F, Bartley D, Bartley Y, Kenyon F (2009) Worm control in sheep in the future. Small Ruminant Research 86:40-45. Janssen M, Weimann C, Gauly M, Erhardt G (2002) Associations between infections with Haemonchus contortus and genetic markers on ovine chromosome 20. 7TH WORLD CONGRESS ON GENETICS APPLIED TO LIVESTOCK PRODUCTION Montpellier, France. Kahn LP (2003) Regulation of the resistance and resilience of periparturient ewes to infection with gastrointestinal nematode parasites by dietary supplementation. Australian Journal of Experimental Agriculture 43:1477-1485. Karlsson LJ, Greeff JC (2006) Selection response in fecal worm egg counts in the Rylington Merino parasite resistant flock. Australian Journal of Experimental Agriculture 46:809-11. Kemper KE, Palmer DG, Liu SM, Greeff JC, Bishop SC and Karlsson LJE (2010) Reduction of faecal worm egg count, worm numbers and worm fecundity in sheep selected for worm resistance following artificial infection with Teladorsagia circumcincta and Trichostrongylus colubriformis. Veterinary Parasitology 171:238-246. Kemper KE, Emery DL, Bishop SC, Oddy H, Hayes BJ, Dominik S, Henshall JM, Goddard ME (2011) The distribution of SNP marker effects for faecal worm egg count in sheep, and the feasibility of using these markers to predict genetic merit for resistance to worm infections. Genetic Research 93:203-219. 21 Kijas JW, Lenstra JA, Hayes B, Boitard S, Neto LR, San Cristobal M, Servin B, McCulloch R, Whan V, Gietzen K, Paiva S (2012) Genome-wide analysis of the world's sheep breeds reveals high levels of historic mixture and strong recent selection. PLoS biology10(2):e1001258. Lôbo RN, Vieira LS, Oliveira AAD, Muniz EN, Silva JMD (2009) Genetic parameters for faecal egg count, packed-cell volume and body-weight in Santa Inês lambs. Genetics and Molecular Biology 32:228-294. Lôbo RNB, Pereira IDC, Facó O, McManus CM (2011) Economic values for production traits of Morada Nova meat sheep in a pasture based production system in semi-arid Brazil. Small Ruminant Research 96:93-100. Matika O, Nyoni S, van Wyk JB, Erasmus GJ, Baker RL (2003) Resistance of Sabi and Dorper ewes to gastro-intestinal nematode infections in an African semi-arid environment. Small Ruminant Reserach 47:95-102. McManus C, Louvandini H, Paiva SR, Oliveira AA, Azevedo HC, Melo CB (2009) Genetic factors of sheep affecting gastrointestinal parasite infections in the Distrito Federal, Brazil. Veterinary Parasitology 166:308-313. McManus C, Facó O, Shiotsuki L, Rolo JLJP, Peripolli V (2019) Pedigree analysis of Brazilian Morada Nova hair sheep. Small Ruminant Research 170:37-42. Mederos AE, Ramos Z, Banchero GE (2014) First report of monepantel Haemonchus contortus resistance on sheep farms in Uruguay. Parasites & Vectors 7:598. Miller JE, Bahirathan M, Lemarie SL, Hembry FG, Kearney MT, Barras SR (1998) Epidemiology of gastrointestinal nematode parsitism in Suffolk and Gulf Coast Native sheep with special emphasis on relative susceptibility to Haemonchus contortus infection. Veterinary Parasitology 74:55-74. Morris CA, Vlassof A, Bisset SA, Baker RL, West CJ, Hurford AP (1997) Responses of Romney sheep to selection for resistance or susceptibility to nematode infection. Animal Science 62:319-329. Morris CA, Wheeler M, Watson TG, Hosking BC, Leathwick DM (2005) Direct and correlated responses to selection for high or low faecal nematode egg count in Perendale sheep. New Zealand Journal of Agricultural Research 48:1-10. Morris CA, Bisset CA, Vlassof A, Wheeler M, West CJ, Devantier BP & Mackay AD (2010) Selecting for resilience in Romney sheep under nematode parasite challenge, 1994-2007. New Zealand Journal of Agricultural Research 53:245-261. Oliveira EJ, Savegnago RP, Freitas LA, Freitas AP, Maia SR, Simili FF, Faro L, Costa RLD, Santana Júnior ML, Paz CCP (2018) Estimates of genetic parameters and cluster analysis for worm resistance and resilience in Santa Inês meat sheep. Pesquisa Agropecuária Brasileira 53:1338-1345. Papadopoulos E, Gallidis E, Ptochos S (2012) Anthelmintic resistance in sheep in Europe: a selected review. Veterinary Parasitology 189:85-88. 22 Perry BD, Randolph TF, McDermott JJ, Sones KR, Thornton PK (2002) Investing in animal health research to alleviate poverty. International Livestock Research Institute (ILRI), Nairobi, Kenya, 148 pp. Rosa GJ (2013) Foundations of animal breeding. In.: Christou P, Savin R, Costa-Pierce BA, Misztal I, Whitelaw CBA (Eds.) Sustainable food production Springer Science + Business Media New York pp. 58-78. Sales N, Love S (2016) Resistance of Haemonchus sp. to monepantel and reduced efficacy of a derquantel/abamectin combination confirmed in sheep in NSW, Australia. Veterinary Parasitology 228:193-6. Sayers G, Good B, Hanrahan JP, Ryan M, Sweeney T (2005) Intron 1 of the interferon γ gene: Its role in nematode resistance in Suffolk and Texel sheep breeds. Research in Veterinary Science 79:191-6. Schwaiger FW, Gostomski D, Stear MJ, Duncan JL, McKellar QA, Epplen JT, Buitkamp (1994) An Ovine Major Histocompatibility Complex DRB1 Allele is Associated with Low Faecal Egg Counts Following Natural, Predominantly Ostertagia circumcincta Infection. International Journal for Parasitology 7:815-822. Scott I, Pomroy WE, Kenyon PR, Smith G, Adlington B, Moss A (2013) Lack of efficacy of monepantel against Teladorsagia circumcincta and Trichostrongylus colubriformis. Veterinary Parasitology. 198:166-71. Snyman MA, Fisher AD (2019) Genetic parameters for traits associated with resistance to Haemonchus contortus in a South African Dohne Merino sheep flock. Small Ruminant Research 176:76-88. Toledo NM (2014) Estudo da estrutura genética de ovinos localmente adaptados do Brasil por meio de marcadores de base única (SNP – Single Nucleotide Polymorphism). 88 pp. Thesis (Master of Animal Science) - Faculty of Agronomy and Veterinary Medicine, University of Brasília. Toscano JHB (2019) Caracterização de respostas imunes locais associadas ao fenótipo de resistência parasitária em cordeiros da raça Morada Nova. 94 pp. Thesis (Master of Animal Science) – Faculty of Agronomy and Veterinary Medicine, São Paulo State University, Jaboticabal. Toscano JHB, Santos IB, Haehling MB, Giraldelo LA, Lopes LG, Silva MH, Figueredo A, Esteves SN and Chagas ACS (2019) Morada Nova sheep breed: resistant or resilient to Haemonchus contortus infection? Veterinary Parasitology X 2:100019. Vanimisetti HB, Andrew SL, Zajac AM, Notter DR (2004) Inheritance of fecal egg count and packed cell volume and their relationship with production traits in sheep infected with Haemonchus contortus. Journal of Animal Science 82:1602-1611. Veríssimo CJ, Niciura SCM, Alberti ALL, Rodrigues CFC, Barbosa CMP, Chiebao DP, Cardoso D, Silva GS, Pereira JR, Margatho LFF, Costa RLD, Nardon RF, Ueno TEH, Curci VCLM, Molento MB (2012) Multidrug and multispecies resistance in sheep flocks from São Paulo state, Brazil.Veterinary Parasitology 187:209-216. 23 Williams AR, Greeff JC, Vercoe PE, Dobson RJ, Karlsson LJE (2010) Merino ewes bred for parasite resistance reduce larval contamination onto pasture during the peri-parturient period. Animal 4:122-127. Wilson T, Wu XY, Juengel JL, Ross IK, Lumsden JM, Lord EA, Dodds KG, Walling GA, McEwan JC, O’Connell AR, McNatty KP (2001) Highly prolific Booroola sheep have a mutation in the intracellular kinase domain of bone morphogenetic protein IB receptor (ALK-6) that is expressed in both oocytes and granulosa cells. Biology of Reproduction 64:1225-1235. Woolaston RR, Piper LR (1996) Selection of Merino sheep for resistance to Haemonchus contortus: genetic variation. Animal Science 62:451-460. Zaros, LG, Neves, MRM, Benvenuti, CL, Navarro, AMC, Sider, LH, Coutinho, LL, Vieira, LS (2014) Response of resistant and susceptible Brazilian Somalis crossbreed sheep naturally infected by Haemonchus contortus. Parasitology Research 113: 1155-1161. Zvinorova PI, Halimani TE, Muchadeyi FC, Matika O, Riggio V, Dzama K (2016) Veterinary Parasitology 225:19-28. 24 CHAPTER 2 – GENETIC PARAMETERS FOR FAECAL EGG COUNT (FEC) PACKED CELL VOLUME (PCV) AND DAILY WEIGHT GAIN IN MORADA NOVA SHEEP INFECTED WITH Haemonchus contortus 2.1. ABSTRACT Gastrointestinal nematodes cause large economic losses in sheep production systems around the world. Selection of animals resistant to infection with GIN has proven to be a feasible and effective approach to address this problem. The aim of this study was to evaluate the possibility of selection for resistance and resilience traits in Morada Nova lambs and to estimate potential correlated responses. A total of 256 lambs and 123 ewes were submitted to two consecutive independent artificial infections with 4,000 infective larvae (L3) of Haemonchus contortus. Records of faecal egg count (FEC), packed cell volume (PCV), and body weight (BW) were taken until day 42 after infection in both challenges. After model definition, estimates of genetic parameters were obtained for individual records as well as for overall traits with repeated measures, using mixed animal models. Phenotypic traits (FEC, PCV and BW/daily weight gain (DWG)) and estimated breeding values (EBVs) of lambs of all three traits were divided by their standard deviations to align variances and, hereafter, a phenotypic as well as a genetic index were established and compared. Heritability estimates for FEC in the first and second parasite challenge were, respectively, 0.25 ± 0.18 and 0.46 ± 0.19 for lambs, and 0.00 ± 0.09 and 0.20 ± 0.16 for ewes. For PCV, heritability estimates were 0.23 ± 0.14 and 0.32 ± 0.16 for lambs and 0.13 ± 0.11 and 0.37 ± 0.18 for ewes. The overall DWG heritability estimate was 0.70 ± 0.21. Genetic correlations of FEC and PCV between lambs and ewes were 0.36 ± 0.08 and 0.42 ± 0.08, respectively. No significant genetic correlation was found between DWG and the other traits, while there was a negative genetic correlation between FEC and PCV (-0.70 ± 0.03). The results of the present study show that selection for low FEC and high PCV is possible in Morada Nova lambs. Selection for low FEC should have a correlated response on PCV (leading to higher PCV), while no correlated response is expected on DWG. Selection for low FEC and high PCV in lambs should lead to low FEC and high PCV in ewes. An index consisting of overall DWG and two records of FEC and PCV (taken 4 to 6 weeks after infection during the second parasite challenge) can be used as selection criterion in the Morada Nova lambs, allowing simultaneous selection for lower FEC, higher PCV and higher DWG. 25 2.2. INTRODUCTION Gastrointestinal nematodes (GIN) restrict the productivity of sheep farming systems worldwide. In Brazilian sheep flocks, haematophagous Haemonchus contortus has the highest clinical and economic importance, causing weight loss, retarded growth, anaemia and deaths (Amarante et al., 2004; Chagas et al., 2013). Control strategies are mainly based on the use of anthelmintics. However, the efficiency of this approach is threatened by the increasing prevalence of drug-resistant parasites (Jackson et al., 2009; Papadopoulos et al., 2012, Veríssimo et al., 2012), and the need for alternative strategies is evident. Selection of animals resistant to GIN infection has proven to be a feasible and effective approach (Bisset et al., 2001; Eady et al., 2003; Kemper et al., 2010), and faecal egg count (FEC), widely used as a measure of resistance, has been included in several commercial breeding programmes (Goldberg et al., 2012; Brown and Fogarty, 2017). Bisset et al. (2001) related that after 21 years of selection for high and low resistance in Romney sheep, the differences in breeding values for log-transformed FEC was 35-fold. Kemper et al. (2010) found that FEC of resistant Rylington Merino sheep was approximately 82% lower compared to unselected animals, after 15 years of selection. Resistant animals are less compromised by parasitized environments and require less anthelmintic treatments, thus lowering costs and increasing the sustainability of production. From the direct impact of selection on FEC, significant reductions in larval contaminations were shown in pastures grazed by lambs selected for high resistance, leading to higher weight gain (Bisset et al., 1997; Bishop and Stear, 2003). Williams et al. (2010) obtained similar results for resistant ewes during the periparturient period. Laurenson et al. (2012) estimated that susceptible lambs can benefit from contemporary grazing with resistant animals, reaching higher body weights (BW) than when grazed separately, while the resistant lamb’s performance is not impaired. Despite these findings, the relationship between resistance and performance traits are not consistent throughout studies (Brown and Fogarty, 2017; Gowane et al., 2019; Snyman and Fisher, 2019). According to Bishop and Stear (2003), correlations between resistance and performance traits depend highly on larval contamination level of pastures, nutrition (especially dietary protein) and other environmental factors. Therefore, it is indispensable to evaluate correlations individually for each particular situation. Packed cell volume (PCV) is an interesting trait due to the haematophagia of H. contortus. It depends on the level of infection, which is why it has been defined as a resistance trait (Albers et al., 1987), although there are mechanisms that allow some 26 animals to maintain high PCV despite high infection levels, leading PCV to be considered a resilience trait by most authors (Baker et al., 2003; Bishop, 2012; Aguerre et al., 2018). Selection for high PCV or other indicators of anaemia is recommended in order to capture these mechanisms, although the concomitant selection for low FEC is indicated in many situations, especially in breeds that are not highly resistant to parasites (Baker et al., 2003; Bishop, 2012; Snyman and Fisher, 2019). Many studies have related that tropically adapted sheep breeds are more resistant and resilient to GIN infections compared to commercial breeds (Baker and Gray, 2004). Native breeds have generally been subject to little or no artificial selection, which is why they are frequently seen as “unproductive”. However, genotype by environment interactions can occur, with the adapted breeds showing better performance than commercial breeds under high parasite challenge, as evidenced by Baker et al. (2004), who compared the adapted Red Maasai breed (resistant) with Dorper sheep (susceptible). Adapted breeds are an important genetic resource for profitable and sustainable production in tropical environments, and their utilization (with concomitant selection for performance traits) is considered preferable to the introduction of commercial breeds in many situations (Baker and Gray, 2004; Bishop, 2012). Morada Nova is a naturalized Brazilian hair sheep breed that is highly resistant and resilient to GIN infections (Issakowicz et al., 2016; Toscano et al., 2019). This breed is characterized by its small size, high adaptation to tropical climate, high prolificacy, non- existent reproductive seasonality, good maternal ability and excellent pelt quality, but also by low weight gain and carcass quality (Facó et al., 2008; Lôbo et al., 2011). In the present study, genetic parameters for FEC, PCV and weight gain were estimated in a Morada Nova sheep flock after artificial infection with H. contortus in order to investigate the possibility of selection for resistance, and to evaluate the relationship between resistance and performance in lambs. 2.3. MATERIAL AND METHODS 2.3.1. Experimental animals and phenotypes The experiment was conducted in a Morada Nova flock at Embrapa Pecuária Sudeste (CPPSE), São Paulo state, Brazil. The location possesses tropical climate, with an annual dry season typically occuring between May and September. A total of 123 ewes were mated to 4 rams in two consecutive years, obtaining 256 lambs, born between April 27 and May of 2017 and March and May of 2018. In each year, two groups of animals were formed according to their birth date, with animals of the same group being weaned and submitted to further experimentation procedures together. At approximately 100 days of age, lambs were weaned, records of FEC, PCV and body weight (BW) were taken and animals were drenched using monepantel (Zolvix®, Novartis, 2.5 mg/kg BW) in order to eliminate natural infection. Subsequently, lambs were separated by sex and transferred to new paddocks. In September of 2018, all ewes were also drenched with monepantel and records of FEC and PCV were taken on the same day. Fifteen days after the first drench, lambs and ewes were infected with 4,000 infective larvae (L3) of Haemonchus contortus. FEC, PCV and body weight were monitored regularly until day 42 after infection, when animals were drenched again in order to end the first artificial infection. Fifteen days after the end of the first trial, the second trial was iniciated, with infection and sampling following the same protocol that was used for the first trial. Management practices have been discussed in detail by Toscano et al. (2019). Eight records of FEC, 6 records of PCV and 4 records of BW were thus obtained under artificial challenge, with samples of FEC being taken on day 21, 28, 35 and 42 after each infection (FECd21-1, FECd28-1, FECd35-2 and FECd42-1 during first, and FECd21- 2. FECd28-2, FECd35-2 and FECd42-2 during second parasite challenge). For PCV, samples were taken on day 14, 28 and 42 after infection (PCVd14-1, PCVd28-1 and PCVd42-1 during first, and PCVd14-2, PCVd28-2 and PCVd42-2 during second infection). For BW, the sampling days were day 28 and 42 after challenge (BWd28-1, BWd42-1, BWd28-2 and BWd42-2). Records of FEC, PCV and BW were taken at weaning, under natural infection (FECweaning, PCVweaning, Bwweaning). For FEC, PCV and BW, analyses were conducted separately for each sampling date. For FEC and PCV, additional analyses were conducted for the overall trait, including all records taken during both parasite challenges (AllRepFEC, AllRepPCV), as well as for each parasite challenge, using data of all records taken during first or second challenge (RepFEC1, RepFEC2, RepPCV1, RepPCV2) After the recording of phenotypes, lambs were ranked based on their FEC values and categorized into phenotypic groups of susceptible (20%), intermediate (60%) and resistant (20%) animals. Further analyses of these phenotypic groups have been conducted and described by Toscano et al. (2019). 28 2.3.2. Statistical analysis Phenotypic data of FEC and PCV from 256 lambs and 123 ewes were used for analyses, while body weight (BW) and weight gain were only accessed in the lambs. The ewes’ BWs remained relatively constant as they were adult animals, not in a growing phase. The distributions of FEC data were found to be positively skewed, which is why a log-transformation (log10(FEC+25)) was performed in order to achieve more symmetrical distributions. Daily weight gain (DWG) was calculated in lambs for the period between weaning and the last day (day 42) of the second challenge (totalDWG), and for each parasite challenge (DWG1, DWG2), between days 0 and 42. In order to define the models of the genetic analyses, fixed effects were tested using the "R" software (R Core Team, 2018), applying a significance level of 0.05. For lambs, fixed effects of sex, group (2 age g