Impact of reproductive biotechnologies on genetic variability of Argentine Polo horses

Manuscript Details Manuscript number LIVSCI_2019_317_R1 Title Impact of reproductive biotechnologies on genetic variability of Argentine Polo horses Article type Research Paper Abstract The Argentine Polo Horse (AP), an autochthonous breed officially created in Argentina in the early 1980s, is globally recognized as the best equid for playing polo. Their breeding is characterized by the use of cutting-edge assisted reproductive techniques such as large-scale embryo transfer (ET) programs and cloning. The aim of this study was to determine the impact of the use of reproductive biotechnologies on the genetic structure, variability and reproductive parameters of the AP breed using genealogical data (81,633 pedigree records). In total, 18,077 animals were drawn from the last generation (2006-2015) to be employed as the reference population (WP), which was further divided into two subsets: animals produced (ET; n=13,478) and not produced by ET (NOT-ET; n=4,599). Horses produced by ET showed a significant decrease in generation interval compared with NOT-ET. Similarly, the number of stallions and broodmares, the number of foals per stallion and broodmare as well as inbreeding (F=0.89%) and average relatedness (AR=1%) were higher in ET compared with NOT-ET (F=0.6%; AR=0.54%). Our analysis also revealed that the effective number of founders and ancestors in ET showed a disproportionate gene contribution and a strong genetic bottleneck. Furthermore, the inter-herd fixation index (FST) revealed an increased genetic flow between herds and higher internal relatedness values within ET horses. In conclusion, the use of large-scale ET programs decreased genetic variability (lower effective population size and number of founders and ancestors and higher F, AR and coancestry), increased the genetic flow among herds and decreased the generation interval, thereby contributing to the higher rates of genetic progress in the AP. Keywords Embryo transfer; Reproductive biotechnology; Inbreeding; Pedigree information Corresponding Author Florencia Azcona Corresponding Author's Institution Instituto de Genética Veterinaria "Ing. Fernando Noel Dulout" Order of Authors Florencia Azcona, Mercedes Valera, Antonio Molina, Pablo Trigo, Pilar Peral García, Marina Solé, Sebastián Demyda Peyrás Suggested reviewers Rute Santos, María José Sánchez Guerrero Submission Files Included in this PDF File Name [File Type] Reviewers letter.docx [Response to Reviewers] Checklist.docx [Revision Checklist] Highlights.docx [Highlights] Main Manuscript.docx [Manuscript File] Fig. 1.pdf [Figure] Fig. 2.pdf [Figure] Fig. 3.pdf [Figure] Conflict of interest.docx [Conflict of Interest] To view all the submission files, including those not included in the PDF, click on the manuscript title on your EVISE Homepage, then click 'Download zip file'. 1Highlights 2More than 60% of AP were produced by embryo transfer (ET). 3F, AR, and coancestry were higher in horses born by ET, while Fe and Fa were 4lower. 5ET also produced a greater reduction in the genetic variability of the individuals. 6ET rose genetic flow and reduced the generation interval, increasing the genetic 7progress. 1 1Impact of reproductive biotechnologies on genetic variability of Argentine Polo horses 2 3Florencia Azconaa*; Mercedes Valerab; Antonio Molinac; Pablo Trigoa; Pilar Peral Garcíaa; 4Marina Soléd and Sebastián Demyda-Peyrása* 5 6aIGEVET – Instituto de Genética Veterinaria "Ing. Fernando N. Dulout" (UNLP - CONICET 7LA PLATA). Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata. Calle 60 y 8118 s/n, 1900-La Plata, Argentina. 9bDepartamento de Ciencias Agroforestales. Escuela Técnica Superior de Ingeniería 10 Agronómica. Universidad de Sevilla. Ctra. de Utrera km 1. 41013-Sevilla, España. 11 c Departamento de Genética; Universidad de Córdoba, CN IV KM 396 Edificio Gregor Mendel, 12 14007; Córdoba, España 13 dUnit Animal Genomics, GIGA-R & Faculty of Veterinary Medicine, University of Liège – B34 14 (+1), Avenue de l’Hôpital 1, 4000-Liège, Belgium. 15 16 *Co-corresponding authors 17 Phone: +54 221 4211799 18 Florencia Azcona: [email protected] 19 Sebastián Demyda Peyrás: [email protected] 20 21 Short title: Genetic variability of Argentine Polo horses 2 22 Abstract 23 The Argentine Polo Horse (AP), an autochthonous breed officially created in Argentina in the 24 early 1980s, is globally recognized as the best equid for playing polo. Their breeding is 25 characterized by the use of cutting-edge assisted reproductive techniques such as large-scale 26 embryo transfer (ET) programs and cloning. The aim of this study was to determine the impact 27 of the use of reproductive biotechnologies on the genetic structure, variability and reproductive 28 parameters of the AP breed using genealogical data (81,633 pedigree records). In total, 18,077 29 animals were drawn from the last generation (2006-2015) to be employed as the reference 30 population (WP), which was further divided into two subsets: animals produced (ET; n=13,478) 31 and not produced by ET (NOT-ET; n=4,599). Horses produced by ET showed a significant 32 decrease in generation interval compared with NOT-ET. Similarly, the number of stallions and 33 broodmares, the number of foals per stallion and broodmare as well as inbreeding (F=0.89%) 34 and average relatedness (AR=1%) were higher in ET compared with NOT-ET (F=0.6%; 35 AR=0.54%). Our analysis also revealed that the effective number of founders and ancestors in 36 ET showed a disproportionate gene contribution and a strong genetic bottleneck. Furthermore, 37 the inter-herd fixation index (FST) revealed an increased genetic flow between herds and higher 38 internal relatedness values within ET horses. In conclusion, the use of large-scale ET programs 39 decreased genetic variability (lower effective population size and number of founders and 40 ancestors and higher F, AR and coancestry), increased the genetic flow among herds and 41 decreased the generation interval, thereby contributing to the higher rates of genetic progress in 42 the AP. 43 44 Keywords: Embryo transfer; Reproductive biotechnology; Inbreeding; Pedigree information; 3 45 1. Introduction 46 The analysis of the genetic and demographic structure of animal populations is a valuable 47 tool to highlight important circumstances affecting their history (Valera et al., 2005). This 48 information has also been useful to analyze the dynamic changes in gene pools during a certain 49 period. As example, these methodologies have been used to describe the genetic structure of 50 horse breeds and populations worldwide (Maccluer et al., 1983; Poncet et al., 2006; Bartolomé 51 et al., 2011; Pjontek et al., 2012; Vicente et al., 2012; Borowska and Szwaczkowski, 2014; 52 Petersen et al., 2014). Furthermore, these results are particularly interesting as an auxiliary tool 53 in breeding decisions since in this species, management and mating policies are normally 54 decided by breeders in an artisanal way (Medeiros et al., 2014; Giontella et al., 2018). However, 55 the existence of detailed and reliable pedigree information is the first and necessary step to 56 obtain reliable results as well as for the implementation of breeding programs based on more 57 scientific methodologies (Cervantes et al., 2008). 58 The Argentine Polo horse (AP) is a popular horse breed among elite polo players (Maserati 59 and Mutto, 2016). The breed was officially created in the early 1980s and is managed by the 60 Asociación Argentina de Criadores de Caballos de Polo (AACCP), which is responsible for 61 determining the policies and rules for breeding practices. Its origin was associated mainly to 62 the crossing of elite native crossbred with Thoroughbred horses; however, the incorporation of 63 individuals of any breed that met certain criteria of aptitude and type was also allowed, a policy 64 that remains until the present time. Interestingly, since its creation in 1984, the AACCP 65 permitted, and even encouraged, the use of any assisted reproductive technique available for 66 the species. Thus, AP breeding is characterized by the existence of massive embryo transfer 67 (ET) programs and the use of state-of-the-art reproductive techniques such as the production of 68 sex-selected embryos (Herrera et al., 2014), oocyte transfer (Riera et al., 2016) and cloned 69 animals (Maserati and Mutto, 2016; Gambini and Maserati, 2018). Consequently, the 70 percentage of ET-produced individuals steadily increased during the last decades, currently 4 71 accounting for more than half of the foals produced, and positioning the AP as a leading breed 72 in terms of reproductive methods and becoming the most exported and worldwide distributed 73 Argentinean horse, with near 1500 individuals sold abroad every year (MinAgri, 2015). This 74 fact also made AP an interesting model to analyze the effect of ET in a commercial horse 75 population. In this sense, it was demonstrated that ET produce an increase in the genetic gain 76 and selection pressure in domestic animals (Mota et al., 2013), but it was also associated to 77 decreases in terms of genetic variability among and within herds. However, and despite the 78 cultural and economic importance of ET in the horse breeding, the association between the use 79 of this assisted reproductive methodology and genetic structure and variations has not yet been 80 assessed. Therefore, the aim of this study was to evaluate the effect of the use of ET programs 81 on reproductive parameters and genetic variability in a commercial horse breed characterized 82 by the use of massive ET programs. 83 84 2. Materials and methods 85 2.1. Genealogical information 86 Pedigree information of the AP was obtained from the AACCP and the Sociedad Rural 87 Argentina public databases. Additional pedigree records were obtained from the Argentine 88 Thoroughbred Studbook in order to reconstruct the registry of founder individuals. The final 89 database included 81,633 horses registered since the AACCP foundation in 1984 until 2015. 90 Only individuals with at least three equivalent complete generations (ECG; according to 91 Maignel et al. (1996)) and belonging to the last generation (born between 2006 and 2015), were 92 used as reference population (WP; n=18,077). This group was further divided into two main 93 subgroups: horses born (ET; n=13,478) and not born (NOT-ET; n=4,599) by embryo transfer. 94 2.2. Demographic analysis 95 The initial analysis included the number of animals registered per year, the number of 96 individuals used as stallions and broodmares, and the female/male rate of births. Generation 5 97 interval (GI), defined as the mean age of the parents at the birth of the offspring which are 98 destined for reproduction (James, 1977), was also determined in the four possible pathways 99 (father-son, father-daughter, mother-son, and mother-daughter). Differences in sex ratio, 100 number of offspring and GI between groups were analyzed using Zand T-test. 101 2.3. Genetic variability parameters 102 Genetic variability of WP was evaluated with the following parameters: 1) Inbreeding 103 coefficient (F), defined as the probability that an individual has two identical alleles by descent 104 (Wright, 1931); 2) Rate of inbreeding between groups (FR), which quantifies the proportion of 105 F of the ET group compared with NOT-ET. In this study, this parameter was computed as: 106 𝐹 𝑅 = 𝐹 𝐸𝑇 ‒ 𝐹 𝑁𝑂𝑇 ‒ 𝐸𝑇 𝐹 𝑁𝑂𝑇 ‒ 𝐸𝑇 107 Where FET and FNOT-ET were the inbreeding coefficients of ET and NOT-ET, respectively, 108 in order to determine the dynamics of the inbreeding variation between groups. 3) Average 109 relatedness (AR), defined as the probability that an allele randomly chosen from the whole 110 population belongs to a given animal (Gutierrez and Goyache, 2005), to determine the 111 representation of a given individual in the total population or the percentage in which a founder 112 was involved in the origin of the breed; 4) Coancestry coefficient for each pair of individuals, 113 estimated as the probability that two individuals share allele identity by descent (Malécot, 114 1948), to determine the average relatedness between animals; and 5) Effective population size, 115 defined as the number of animals that would maintain the current increase in inbreeding if they 116 contributed equitably to the next generation, estimated via individual increase in both 117 inbreeding (Ne) (Gutierrez et al., 2009) and pairwise coancestry (NeC) (Cervantes et al., 2011). 118 Differences between groups were determined using the Mann-Whitney U test since sample data 119 were not normally distributed (Kolmogorov Smirnov test, P<0.01). 120 2.4. Founder and individual gene origin 6 121 The probability of gene origin was determined through the effective number of founders 122 (Fe), defined as the number of founders that could produce the same genetic diversity observed 123 in the population, and the effective number of ancestors (Fa), defined as the minimum number 124 of individuals (not necessarily founders) that explains the genetic variability of the breed. This 125 dual approach was employed to determine the existence of a genetic bottleneck in the 126 population, as was proposed by Boichard et al. (1997), by calculating the Fe/Fa ratio, which is 127 higher than 1 when the contribution of ancestors is unequal. Additionally, we determined the 128 number of founder genome equivalents (Fg), defined as the number of founders that would be 129 expected to produce the same genetic diversity as in the population under study if the founders 130 were equally represented and no loss of alleles occurred (Lacy, 1989). This parameter 131 specifically accounts for the random loss of alleles during genetic bottlenecks produced by the 132 unbalanced contributions of founders. Finally, the breed composition of the ten individuals 133 which contributed the most to the genetic variability of the breed was assessed to determine the 134 influence of foreign breeds in the AP composition. 135 2.5. Population structure 136 The possible structuring in partially isolated subpopulations among breeders was determined 137 with the fixation index (FST), following the methodology described by Caballero and Toro 138 (2000). This parameter was calculated in herds with at least 40 registered individuals belonging 139 to both ET and NOT-ET groups (n=83 herds). Thereafter, FST was estimated among herds which 140 had individuals in both groups in order to obtain the FST Spearman´s rank correlation coefficient 141 between groups (ET and NOT-ET). This parameter allowed to determine if the increase of 142 genetic similarity among herds was affected by the breeding system. To identify less evident 143 subpopulation divisions derived from partial structuring, an additional comparison was 144 performed according to the methodology described by Cervantes et al. (2011) which is based 145 on the comparison between effective population sizes calculated from individual increases in 146 inbreeding and in pairwise coancestry (Ne and NeC). 7 147 All parameter estimations were performed using ENDOG v.4.8 software (Gutierrez and 148 Goyache, 2005). The statistical analysis was carried out with STATISTICA package v.12. 149 (StatSoft., 2012). 150 3. Results 151 3.1. Demographic analysis 152 The number of individuals born and registered annually since the AACCP creation in 1984 153 increased considerably every year until 2009 (Figure 1), with approximately 40% of the total 154 population registered during the last generation (2006-2015). In those years, the female/male 155 ratio was relatively steady (near 1.5:1), but it began to increase by 2010, reaching rates close to 156 4:1 in 2014 (Figure 1). Pedigree completeness was moderate, showing the average maximum 157 and equivalent complete generations traced equal to 11.3 and 4.9, respectively, which are robust 158 values in comparison with similar studies (Bartolomé et al., 2011; Medeiros et al., 2014). 159 Similarly, equivalent complete generation in ET and NOT-ET groups was 5.1 and 4.2, 160 respectively. 161 The number of individuals produced by ET from 1990 to 2015 showed a steady increase 162 since 2004, reaching percentages higher than 60% of the total number of horses enrolled in the 163 AACCP during the last four years studied (Figure 2). The increased female/male ratio was also 164 higher in ET as compared with WP (P<0.01), depicting a clear preference of breeders for 165 producing mares. 166 At the time of this study, only 10% of males and 27% of females from the whole population 167 became stallions and broodmares, producing an average of 22.7 and 3.4 foals per individual, 168 respectively. However, there was a clear imbalance in individual contribution, since only 14 169 stallions (0.6% of the total) registered more than 300 foals each, accounting for 12.5% of the 170 foal crop. Furthermore, a higher percentage of horses was used as stallions in ET (11.7% vs 171 9.84% in NOT-ET; P<0.01), also producing five more foals per individual (26 in ET vs 21 in 172 NOT-ET; P<0.05). On the contrary, the percentage of females used as breeding mares was 14 327 (2000) described that individuals with F > 6.25% (which its number it is also increasing in AP 328 and particularly in ET) could induce inbreeding depression in dairy cattle. Even though average 329 inbreeding is still low in the breed, the increasing trend observed during the last years, 330 particularly in ET-produced horses, is a parameter that could be considered in future mating 331 decisions among the breeders in order to avoid the appearance of inbreeding depression among 332 individuals. 333 The effects produced using ET were also reflected in the average coancestry of each group 334 (significantly higher in ET), mostly due to an increased selection intensity over the mares, 335 which are producing more foals during his reproductive life thus increasing their 336 representativeness in the pedigree, producing an increase in the AR values and further limiting 337 genetic diversity. In order to cope with this situation, the Andalusian Horse Breeder Association 338 has successfully developed and implemented systematical approaches to monitor inbred mating 339 using an ad-hoc software to determine the relationships among all available breeders in a herd 340 (Melgarejo et al., 2000), which decreased substantially the average inbreeding rate in a 341 relatively short lapse of time (Valera et al., 2005). Since the use of ET techniques increases 342 mating possibilities by using stallions or mares which are in different herds and locations, the 343 implementation of this type of technologies could help to avoid the mating highly related 344 animals. 345 Effective population size is an important indicator of the evolution of the genetic variability 346 of a given population. In WP NeC was lower than other composite breeds, such as Brazilian 347 Sport Horse (NeC 188.5, Medeiros et al. (2014)) Anglo-Arab horse (NeC 291.2, Cervantes et al. 348 (2011)) and Spanish Sport Horse (NeC=1046, Bartolomé et al. (2011)), suggesting a reduced 349 variability probably due to the selection strategy employed. Cervantes et al. (2011) also 350 demonstrated that NeC and Ne are expected to present very similar values to each other in a non351 genetically structured population, since individual increase in inbreeding tend to raise by the 352 presence of subdivision in a population, while individual increase in coancestry is practically 15 353 not affected. In our study, the NeC/Ne ratio in NOT-ET was twice than in ET, supporting the idea 354 that the individuals bred by natural gestation were more related within groups, probably due to 355 the lower connection between herds since broodmares normally remains in the same herds 356 during all his reproductive life. This was also shown by the increased FST value in this group as 357 well as by the low correlation among herds. Therefore, the joint analysis of this parameters 358 supports the idea that ET allows an increased mobility of the selected germplasm among herds, 359 mostly via female lineages, in comparison with NOT-ET, in which the homogeneity is observed 360 within rather than between groups. 361 The Fe is another important parameter to determine whether the contribution of the original 362 breeding individuals is balanced. It is expected that the total number of founders is equal to Fe 363 when all of them contribute equally (Lacy, 1989). However, this does not usually occur in 364 livestock populations, where Fe is generally smaller, indicating a loss of genetic diversity in 365 proportion to how those values differ. This fact was evident in several horse populations 366 studied, such as the Spanish Sport Horse (Bartolomé et al., 2011) and the Andalusian Horse 367 (Valera et al., 2005). In our study, Fe and Fa were substantially lower than the number of 368 founders and ancestors in WP, whereas the Fe/Fa ratio suggest a clear bottleneck in the 369 population. This trend was highly exacerbated in ET population, where Fe and Fa were lower, 370 and the bottleneck is more noticeable compared with NOT-ET. This fact could be explained by 371 the use of a reduced number of stallions and broodmares, causing a high selection pressure, 372 which is also supported by the differences observed in the Fg. Since our clustering criteria was 373 only the use of ET techniques during breeding, this study clearly demonstrate, in a realistic 374 breeding situation with than 18,000 horses analyzed, that large ET programs have a marked 375 effect on the reduction of the population genetic variability. 376 Finally, it was noteworthy that the most contributing ancestors of the breed were 377 Thoroughbred horses. Beyond the fact that the incorporation of animals of any origin was 378 allowed, this information confirmed that the AP breed was highly influenced by this breed in 16 379 its beginnings. However, our analysis also showed that the Thoroughbred influence was 380 produced by a scarce number of stallions extensively employed as breeders during a long period 381 of time rather than a by the massive use of Thoroughbred horses as breeders at the beginning. 382 This fact could also have affected genetic variability, as was already described in the Brazilian 383 Sports horse (Medeiros et al., 2014), the Spanish Asturcón (Álvarez et al., 2011), and three 384 Czech draught horse breeds (Vostrá-Vydrová, 2016). In this sense, it is also well known that 385 the Thoroughbred horse is a very old breed created from a few famous stallions (Binns et al., 386 2012) with a limited genetic variability (Cunningham et al., 2001; Bokor et al., 2013). Since 387 this breed was heavily involved in the origins of polo horses, it would not be unwise to assume 388 that such scarce diversity could be dragged towards the AP, further affecting its genetic status. 389 However, this could not be assessed since there are no population studies performed in polo 390 ponies using molecular or genomic data estimations of the genetic variability of the breed. 391 392 5. Conclusions 393 In this study, we analyzed the genealogical data of an AP horse population, recognized 394 worldwide by its sports skills in polo game, in which horses are mainly produced by the use of 395 large-scale ET programs. Our results not only demonstrated that such reproductive 396 methodologies could enhance the distribution of superior germplasm, but also could 397 compromise the genetic variability in a long-term basis. Even though average relatedness and 398 inbreeding have been rising in this population during the last years, the situation is not yet 399 worrying, probably due to the existence of an open studbook. We also demonstrated that 400 inbreeding increase and loss of genetic variability were highly dependent on the use embryo 401 transfer programs. Therefore, their use may be complemented by practices to mitigate the 402 potential negative effect produced by the inbreeding depression on performance traits in the 403 future generations of AP horses. 404 17 405 Acknowledgments 406 We deeply thank the Asociación Argentina de Criadores de Caballos de Polo and particularly 407 MV Guillermo Buchanan, Santiago Ballester and MV Maria Inés Morikawa for data provision 408 and technical assistance. We also thank Hernan Morales Durand by technical assistance in data 409 collection. Thanks are also due to A. Di Maggio for manuscript correction and editing. This 410 study was supported by ANPCyT – FONCyT grant (PICT 2016-0359). Florencia Azcona is a 411 PUE-CONICET fellow (PUE-2016-22920160100004CO). 412 413 Conflict of Interest Statement 414 The authors declare that the research was conducted in the absence of any commercial or 415 financial relationships that could be construed as a potential conflict of interest. 18 416 References 417 Álvarez, I., Royo, L.J., Pérez-Pardal, L., Fernández, I., Lorenzo, L., Goyache, F., 2011. 418 Assessing diversity losses due to selection for coat colour in the endangered bay419 Asturcón pony using microsatellites. Livest. Sci. 135, 199-204. 420 Bartolomé, E., Cervantes, I., Valera, M., Gutiérrez, J.P., 2011. Influence of foreign breeds 421 on the genetic structure of the Spanish Sport Horse population. Livest. Sci. 142, 70422 79. 423 Binns, M.M., Boehler, D.A., Bailey, E., Lear, T.L., Cardwell, J.M., Lambert, D.H., 2012. 424 Inbreeding in the Thoroughbred horse. Anim. 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Evolution of inbreeding coefficient for WP, ET and NOT-ET, in the last generation, and the rate of inbreeding. -50% 0% 50% 100% 150% 200% 0,0% 0,2% 0,4% 0,6% 0,8% 1,0% 1,2% 1,4% 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 Rate of inbreeding Inbreeding coefficient Birth year ET NOT-ET WP Rate of inbreeding Conflict of Interest Statement The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.