Monitoring of genetic diversity in the endangered Martina Franca donkey population 1

Published December 4, 204 Monitoring of genetic diversity in the endangered Martina Franca donkey population R. Rizzi,* E. Tullo,* A. M. Cito, A. Caroli, and E. Pieragostini 2 *Department of Veterinary Science and Technology for Food Safety, University of Milan, 2033 Milan, Italy; Department of Engineering and Management of the Agricultural, Livestock and Forest Systems, University of Bari, 7000 Bari, Italy; and Department of Biomedical Sciences and Biotechnologies, University of Brescia, 2523 Brescia, Italy ABSTRACT: The Martina Franca (MF) donkey, an ancient native breed of Apulia, was mostly famous for mule production. The breed was at serious risk of extinction in the 980s following the decrease in demand for draft animals because they were increasingly replaced by agricultural machinery. Much has been done in the last few decades to safeguard the existing donkey breeds, but the situation remains critical. Successful implementation of conservation measures includes an evaluation of the present degree of breed endangerment, so the aim of this work was to analyze the demographic and genetic parameters of this breed to suggest effective conservation strategies. With a current breed register counting less than 500 recorded animals, the pedigree data set included,658 MF donkeys born between 929 and 2006. Analyses were carried out on the whole data set as well as on a smaller one consisting of 422 living animals. Demographic and genetic variability parameters were evaluated using the ENDOG (v4.6) software. The pedigree completeness level was evaluated as well as the generation length, which was calculated for each of the 4 gametic pathways. This information was obtained from animal birth date records together with those of their fathers and mothers. The effective number of founders (f e ), the effective number of ancestors (f a ), the founder genome (f g ), individual inbreeding (F), average relatedness (AR), and the rate of inbreeding per generation were analyzed to describe the genetic variability of the population. Because pedigree depth and completeness were appropriate, especially regarding the current population, the parameters defining genetic variability, namely, f e, f a, f g, F, and AR, could be reliably estimated. Analysis of these parameters highlighted the endangerment status of the MF donkey. Our special concern was with the increased percentage of males and females exhibiting increased AR values. Moreover, the effective size of the current population, 48.08, is slightly less than the range of the minimum effective size, and the rates of inbreeding per generation found in the current MF population exceed the maximum recommended level of %. Such a scenario heightens concerns over the endangered status of the MF breed and calls for proper conservation measures and breeding strategies, such as selecting individuals for mating when relationships are below 2.5%. Key words: breeding strategy, effective population size, genetic variation, inbreeding, Martina Franca donkey, pedigree analysis 20 American Society of Animal Science. All rights reserved. J. Anim. Sci. 20. 89:304 3 doi:0.2527/jas.200-3379 INTRODUCTION This work was supported by the Apulian Regional Fund ATZ 2008 administered by the Apulian Regional Office for Livestock Management. We are truly grateful to the managerial staff of the Office, F. Nico and G. Ferro, for their aid and support. The authors also thank the director of the Institute for the Improvement of Equid Breeding (Foggia), A. Ursitti, for kindly allowing access to the Institute s data and for his indispensable organizational support, and R. Iliceto for his expert collaboration in collecting the data. 2 Corresponding author: pierelis@agr.uniba.it Received July 30, 200. Accepted December 4, 200. The report on the domesticated donkey in Europe raised concerns over the current rate of extinction of individual breeds as well as of the whole species (Kugler et al., 2008). However, an inversion has recently been noted in the demographics given the growing popularity of donkeys, especially for their use for special health and healthcare needs and recreational opportunities. Domestic animal diversity has received growing attention after the Earth Summit held in Rio de Janeiro in 992. 304

Accordingly, the European Union developed a support system for the sustainable utilization and conservation of farm animal genetic resources, and a series of related policies were instituted in single member countries. In some of them, farm animal genetic resource conservation is carried out within broader rural development and environmental management programs. This is the case of many Italian regions and Apulia, in the southern mainland of Italy, presents 7 native livestock breeds, 4 of which are similarly endangered. One of these is the historical Martina Franca (MF) donkey. The MF donkey meets many of the requirements delineated as important for the implementation of conservation measures to safeguard specific species or breeds (Oldenbroek, 999). There are no doubts as to the historical value of this donkey or its ecological role in the agrosilvopastoral system of the Murgia hills. Its genetic uniqueness is obvious in its very peculiar morphological characteristics stemming from the genetic attitude needed to cope with the enzootic tick-borne pathogens typically found in Apulia. Successful implementation of conservation measures includes evaluation of the present degree of breed endangerment (Gandini et al., 2004), so the aim of this investigation was to analyze the demographic and genetic parameters of the past and current population of the MF donkey breed to recommend effective conservation strategies. MATERIALS AND METHODS Animal Care and Use Committee approval was not requested for this study because the data were obtained from an existing database. The Traditional Breeding Site Apulia has a varied topography ranging from coasts and woods to plains and hills, and the Murgia hill area is a wide calcareous high plain in its geometrical center. Their soil and climate have made the Murgia hills a privileged region for breeding large animals since time immemorial, and the rural area surrounding the town of Martina Franca has always been the traditional breeding site of 2 historical native equid breeds: the Murgese horse and the MF donkey. The Breed The MF donkey is a mesomorphic donkey breed characterized by sturdiness, a dark bay coat, and long furry ears, which make it an attractive animal. With its height ranging from 35 to 60 cm, the MF donkey may be considered a giant of the species. Frugal and with a lively temperament, this donkey can adapt to difficult and rocky ground and may be the best choice for agricultural work in national parks where machinery is banned. Genetic diversity in the Martina Franca donkey Due to its unusual height, the MF donkey was once especially appreciated for mule production, but when draft animal power was replaced by agricultural machinery after World War II mule production progressively decreased. In 948 the MF Donkey and the Murgese Horse Breeders Association was founded. In 98, the Apulian Regional Government (Decision No. 244/8) set up the Centre for the Conservation and Safeguard of the MF donkey at the Russoli Farmhouse near the town of Martina Franca. Thanks to the Regional Office for Livestock Management that has supported MF donkey breeders and related research activities at the Universities of Bari and Teramo in the last few decades, the numerical trend of the population progressively changed. Current Population and Breeding Systems The current donkey population is distributed in almost 45 private farms and a couple of public institutions. Apart from the group of 5 jennies and jackass currently managed at University of Teramo s Faculty of Veterinary Medicine (Carluccio et al., 2008; Contri et al., 200), the biggest breeding nucleus, consisting of 44 jennies and 34 jackasses, is managed by the Apulian Regional Administration. The 2 jacks kept at the Institute for the Improvement of Equid Breeding in Foggia are transferred during the breeding season to farms offering stallion station services. Forty-four females and the other 22 jacks are kept in the Centre for the Conservation and Safeguard of the MF donkey at the Russoli Farmhouse, where they are managed under extensive breeding conditions. The foaling period of the MF donkeys typically ranges from February to early June. Foals are weaned at 5 to 6 mo of age and kept in groups with other similarly aged donkeys. Foals from registered MF parents are monitored since the time of conception when an approved stallion covers the jenny. Their birth is recorded, a hair sample taken, and a microchip implanted (Bramante and Pieragostini, 2005). Data Set and Software Program 305 The pedigree records of MF donkeys are kept at the Centre for the Improvement of Equid Breeding in Foggia, where the MF Donkey Demographic Registry is kept. Records contain the name and identification code of the animal, dam and sire, sex, year of birth, and dead/alive status. The edited pedigree database includes,658 donkeys (668 males and 990 females) born between 929 and 2006. The year of birth of 2 animals was missing and thus derived from the first letter of their name, corresponding to a specific year as envisaged by the MF Donkey Registry criteria, and from the birth year of their first offspring, if present. Pedigrees of jacks and jennies were traced back to ancestors. The analysis was carried out on the whole data set

306 described above (whole population) and on a smaller data set (current population) consisting of 422 living animals (68 males and 354 females) born after 980, registered and used for reproduction. Demographic and genetic variability parameters were evaluated by using ENDOG (v4.6) software (Gutiérrez and Goyache, 2005) based on the 2 data sets with the 2 missing birth dates. The estimated birth dates were considered to describe the trend of some parameters over the years. Pedigree Completeness The pedigree completeness level was evaluated by means of the following parameters: ) the maximum number of generations traced (i.e., the number of generations separating an individual from its furthest ancestor); 2) the number of full traced generations (i.e., the furthest generation with the 2 ancestors known); 3) nj the number of equivalent generations, equal to, g i= ij 2 where n j is the total number of ancestors of the animal j and g ij is the number of generations between j and its ancestor i (Boichard et al., 997); and 4) the proportion of parents, grandparents, and great-grandparents known. Generation Intervals Generation length (i.e., the average age of parents at the birth of offspring kept for reproduction) was calculated for each of the 4 gametic pathways: sire to son, sire to daughter, mother to son, and mother to daughter. These parameters were obtained from records of birth dates of single animals together with those of their fathers and mothers. Rizzi et al. f a = f 2 p k k=, where p k is the marginal contribution of an ancestor k [i.e., the contribution not yet explained by the other ancestors (Boichard et al., 997)]; 3) the effective number of founder genomes (f g ), defined as the number of equally contributing founders with no loss of founder alleles that would be expected to produce the same amount of diversity as in the reference population (Lacy, 989); it was obtained by the inverse of twice the average coancestry of the individuals within the population (Caballero and Toro, 2000); 4) individual inbreeding (F), defined as the likelihood that an individual has 2 identical alleles by descent; it is computed according to Meuwissen and Luo (992); 5) the average relatedness (AR) of each individual, defined as the probability that an allele randomly chosen from the whole population belongs to a given animal; it is computed using the algorithm proposed by Gutiérrez and Goyache (2005); 6) the rate of inbreeding ( DF ) for the generation computed as suggested by Gutiérrez et al. (2008) by averaging the individual increase in inbreeding defined as F = F, where t is the t number of equivalent generations and F is the inbreeding coefficient; and 7) the mean effective population size ( N e ) calculated as Ne =. (2 F ) Breeding Strategy Genetic Variation Measurements To describe the genetic variability of the population the following parameters were analyzed: ) the effective number of founders (f e ), defined as the number of equally contributing founders that would be expected to produce the same genetic diversity as that observed in the population under study (Lacy, 989); it is computed as f e =, where q k is the probability of gene f 2 q k= k origin of the kth founder and f is the real number of founders; 2) the effective number of ancestors (f a ), that is the minimum number of ancestors (founders or not) necessary to explain the complete genetic diversity, determined by the following formula: The maximum limit of relationship coefficient between mated animals was assessed to maintain ΔF in a generation equal or below %. This corresponds to an effective size of 50 animals, which is the level below which fitness of a population steadily decreases (Meuwissen and Woolliams, 994). We considered 5 mating groups in which the relationship coefficient between mated animals was kept below 8, 0, 2.5, 5, and 7.5%. The 8% threshold was set because it is the least feasible limit if all possible matings among all 422 living animals are considered. The Minbreed software (Gandini and De Filippi, 998) was used to identify matings between animals within the limits of relationship reported above. Matings with a relationship coefficient below 8, 0, 2.5, 5, and 7.5% were 6,89, 6,987, 8,303, 0,49, and 2,433, respectively. The inbreeding coefficient (F) for the offspring of each mating was calculated as one-half of the parental relationship coefficient. The inbreeding rate was estimated as described by Gutiérrez et al. (2009) by means

Genetic diversity in the Martina Franca donkey 307 Table. Pedigree completeness both in the whole and the current population Item Whole population Current population Maximum No. of generations traced, mean ± SD 4.67 ± 2.9 6.64 ± 2.90 Maximum No. of complete generations, mean ± SD.97 ±.25 2.60 ±.39 No. of complete equivalent generations, mean ± SD 3.0 ±.83 4.7 ±.9 Percentage of known ancestors from the following: First generation (parents) 88 86 Second generation 72 82 Third generation 54 78 Fourth generation 40 70 Fifth generation 27 55 t of the following formula: F = F, where t is the number of equivalent generations and F is the inbreeding coefficient. Within each group, 70 random matings were selected on the basis of the number of births in 2006 (67 births) and on the assumption of foal per jenny using the SURVEYSELECT procedure (SAS Inst. Inc., Cary, NC). Thirty replicates with this structure were analyzed, and for each replicate the realized effective population size was calculated as Ne = (Gutiérrez et al., 2008) from the overall 2 F mean of inbreeding rate ( DF ) computed by averaging the ΔF i values of the offspring selected. The SE of N e is s = 2 2 N s with N being the number of individuals in the sample, σ ΔF the SD of Δ, and s N e F e N Ne the SE of N e (Gutiérrez et al., 2008). RESULTS AND DISCUSSION Pedigree Completeness and Generation Intervals The mean and SD of the maximum number of traced, complete, and equivalent generations and the proportion of known ancestors through 5 generations are reported in Table. The maximum number of generations separating an individual from its furthest ancestors ranged from 0 (founders) to, but the average number of complete (.97 ±.25) and equivalent generations (3.0 ±.83) indicated that ancestors from the second generation had often been recorded inaccurately in the whole population. In living animals the average number of traced, complete, and equivalent generations increased to 6.64 (±2.9), 2.60 (±.39), and 4.7 (±.9), respectively. The average generation interval for the whole population was 8.86 yr and 9.09 yr for the current population. In particular, the generation interval was longer in the sire-offspring pathways than in the dam-offspring pathway (Table 2). It is worth noting, however, that the greater value of 0.32 yr relative to the dam-son pathway was probably due to the 8 jennies whose age at delivery of the offspring included was over 20 yr, whereas only 2 males were 20 yr old at the birth of their offspring. Pedigree completeness is a crucial point in the genetic management of an animal population. Individuals with unknown parents and their offspring are generally assigned a zero inbreeding coefficient even if they are somehow related. If the pedigree contains a large number of missing parents, the inbreeding trend in a population could be underestimated, thus delaying proper actions to decrease inbreeding (Lutaaya et al., 999; Cassell et al., 2003). In other donkey populations, pedigrees were not as deep-rooted as in MF donkey. In the Amiata donkey the greatest number of traced generations was 4 and the average maximum, complete, and equivalent generations were.4, 0.53, and 0.78, respectively (Cecchi et al., 2006). Similarly, for the Catalonian donkey breeds and its subpopulations, the numbers of complete generations ranged from 0.8 to.83, whereas those of the equivalent generations ranged from.2 to 2.78 (Gutiérrez et al., 2005). Table 2. Generation interval (mean and SD) for the 4 parent-offspring pathways, for the whole and the current population Whole population Current population Pathway n Years n Years Sire-son 67 9.24 ± 4.9 9 9.95 ± 4.88 Sire-daughter 387 9.2 ± 3.90 42 9.08 ± 4.03 Dam-son 67 8.64 ± 3.93 9 0.32 ± 4.58 Dam-daughter 389 8.58 ± 4.9 43 8.82 ± 4.29

308 Rizzi et al. Table 3. Parameters of genetic variability in the Martina Franca donkey Item Whole population Current population Number of contributing founders (f) 33 20 Number of contributing ancestors 33 08 Effective number of founders (f e ) 22 9 Effective number of ancestors (f a ) 8 3 Founder genome equivalent (f g ) 4 7 Number of ancestors contributing to 50% of genetic variability 7 5 F, % 3.95 6.87 AR, % 7.35 9.80 DF, % 0.95.04 N e 53 48 F = individual inbreeding; AR = average relatedness; DF = rate of inbreeding; N e = effective population size. In the first generation of the MF donkey, the percentage of known ancestors in the whole population was greater (88%) than in the current population (86%), indicating that in the 960s there was a large number of registered individuals. In the following generations, the pedigree completeness of the whole population presented a pattern substantially concordant with the results reported for the Catalonian donkeys, where the proportion of known ancestors in the fifth generation was less than 20% (Folch and Jordana, 998). Our results for the equivalent generations and the proportions of known ancestors in the first 5 generations reflect the pedigree information of the living animals and are comparable with the ones reported in 3 French cattle breeds (Boichard et al., 997) and in Canadian Holsteins (Van Doormaal et al., 2005). The good quality of the MF donkey pedigree determined a high level of confidence in the subsequent estimation of genetic variation parameters. In the Catalonian and Amiata donkeys, the average generation intervals found (6.74 and 6.65 yr, respectively) were shorter than in the MF donkey (Folch and Jordana, 998; Cecchi et al., 2006). The long generation interval for the MF donkey may be mainly ascribed to the slow turnover rate because the most favored and popular sires and dams continued to contribute progeny to the next generation for years. Prolonging the generation interval may be a method to increase the number of sires and dams selected for breeding, thereby incrementally increasing the effective population size, which is inversely proportional to the rate of inbreeding (Meuwissen, 999). Genetic Variation Measurements The results for the probability of gene origin, inbreeding, and average relatedness both in the whole and in the current population are shown in Table 3. The number of founders, namely individuals with unknown parents, was 9, but only 33 provided their genetic contribution to the whole population, and only 20 out of these contributed to the current population. Preservation of genetic diversity from founder animals is measured by the f e that indicates the number of equally contributing founders that would be expected to produce the same genetic diversity as in the population under study (Lacy, 989); thus, the f e is large, even if the expected contributions of founders are balanced (Boichard et al., 997). The ratio between the f e and the f (f e /f), obtained analyzing the whole and the smaller MF donkey data set, was again 0.6, suggesting that the genetic information from 5 out of 6 founders must be considered lost. Given the magnitude of the f e and the f e /f, it may be assumed that in the MF donkey the frequent use of only a few individuals for breeding led to a loss of genetic variability. This assumption is confirmed by the small number of ancestors contributing to 50% of genetic variability, namely 7 for the whole population and 5 for the current population. In the Catalonian and Amiata donkeys the f e was greater than the one found for the MF donkey (Gutiérrez et al., 2005; Cecchi et al., 2006), but the decreased level of pedigree completeness of those populations may have caused an overestimation of these parameters, as suggested by Boichard et al. (997). Thus, even the large number (8) of founders explaining the 50% of genetic variability in the Amiata donkey may be due to the decreased pedigree depth. The f a is defined as the minimum number of ancestors necessary to explain the complete genetic diversity of a population and considers the contribution of an ancestor not yet accounted for by other ancestors. The f a supplements f e, which is often overestimated and does not account for the possible bottleneck in a population (Boichard et al., 997). The difference between f e and f a suggested a decrease in the genetic variation of the MF donkey due to the documented bottleneck the breed experienced in the 980s and confirmed by the increase in the number of newborns in this period (Figure ). Unlike f a, the founder genome equivalent (f g ), accounts for additional random losses of genes during segregations and describes the amount of founder variation in a population more accurately (Caballero and Toro, 2000). The degree to which f g is smaller indicates

Genetic diversity in the Martina Franca donkey 309 Figure. Trends in level of inbreeding (F), average relatedness (AR), and number of individuals born (N) by year of birth in the period 929 to 2006. the degree of random loss of alleles. In MF donkeys the loss of genetic variation is confirmed by the values of f g (4 and 7), and the decrease in f e is larger in the current population. In particular, the value recorded in the whole population (64%) was greater than that of the current population (38%). The reduced f g in the current population is due to the greater average inbreeding as well as to the smaller number of individuals. Average inbreeding and AR were less in the whole population than in the current one (Table 3), showing an increasing trend throughout the years (Figure ). The AR parameter may be used as a complement of F in predicting the long-term inbreeding of a population and to make changes in the genetic management of a population. Random mating AR in a generation is twice the expected average inbreeding coefficient in the next one. However, in our sample AR increased throughout the years, showing that breeders mated more related individuals, especially from 970 to 990 when the number of animals dramatically decreased and AR was more than 2-fold the value of F. Very small values of mean F and AR (i.e., 0.0029 and 0.0094) were reported for the Amiata donkey, which, as mentioned above, presented a shallow and incomplete pedigree. By contrast, Gutiérrez et al. (2005) reported greater F and AR coefficients in the living population of Catalonian donkeys (F = 0.047 and AR = 0.046) and overall in the Berga subpopulation (F = 0.072 and AR = 0.066). The values found in the current MF population were very similar to those reported for the Berga subpopulation, but in Berga these parameters seem to come from the attempts to obtain a highly selected and morphologically homogenous herd, whereas in the MF donkey the greater level of inbreeding is to be ascribed to the lack of genetic management and to the bad breeding practice of mating only a few related animals. The rates of inbreeding per generation and the related N e reflect the estimates of AR and F. The ΔF found in the current MF donkey population exceeded the recommended maximum levels of ΔF (%) and N e (50) to maintain genetic variation and fitness in a population. By contrast, the results obtained in the whole population were ΔF = 0.9% and N e = 52.6. Breeding Strategy Figure 2 shows the dispersal of the realized effective population size for each sample obtained when the relationship coefficients between mated animals are maintained below 8, 0, 2.5, 5, and 7.5%. When matings between individuals with relationship coefficients less than 2.5% are considered, all replicates present a mean effective population size greater than 50 and ranging from 55. (±3.) to 77.5 (±6.9). The results highlighted that using animals with relationships less than 2.5% can help minimize the inbreeding rate and increase the effective population number. Breeders should make efforts to achieve maximum 0% relationships in matings to ensure an effective population size of 00 in generation and abate the risk of extinction for MF donkeys. The number of possible matings with relationships below 0% (6,987) makes this a feasible aim. As outlined in the Convention on Biological Diversity (CBD, 992), in situ conservation must be considered as a priority for the safeguard of livestock breeds (Gandini et al., 2004). For this to occur, economic sustainability is a prerequisite. At present, this does not clearly seem to be an issue as far as the MF donkey is concerned. The current demand for the MF donkey is fortunately on the rise thanks to the growing number of holiday or educational farms (or both) in Italy, where the qualities of this breed are highly appreciated. Gandini and Villa (2003) underscored the importance of the role of certain breeds in nurturing and perpetuating local traditions. Because of its presence since ancient times and its role in the agricultural system and especially in the Murgia landscape, the MF donkey has recently been the focus of growing attention from the local population in order to restore the common values of Apulian culture and tradition. However, once the risk of breeders discarding the MF donkey has been partly overcome, the first major set of concerns for the conservation of the breed

30 Rizzi et al. Table 4. Percentage distribution of males and females in the current Martina Franca donkey population according to inbreeding value (F) and average relatedness (AR) F AR Probability Females, % Males, % Females, % Males, % 0 25.42 0.29 >0 and /6 28.25 7.65 22.32 8.82 >/6 and 2/6 32.49 57.35 46.6 47.06 >2/6 and 3/6 8.76 3.24 3.07 44.2 >3/6 and 4/6 3.67 >4/6 and 5/6.4.47 regards its biological sustainability. It is generally acknowledged that understanding the origin and subsequent history and evolution of a breed is essential to the design of sustainable conservation and utilization strategies. Our study tracked almost 80 yr of the genetic history of the MF population. The level of pedigree depth and completeness was good, especially in the living population, and permitted the reliable estimation of the parameters defining genetic variability, such as f e, f a, founder genome equivalent, inbreeding, and relatedness. Analysis of these parameters highlighted that part of the genetic variability deriving from founders was lost in the course of years, and especially made clear that the threat of extinction still looms over the MF donkey. Our concern is with the increased percentage of males and females exhibiting increased AR values (Table 4). Moreover, the effective size of the current population, numbering 48.08, is slightly less than the range of the minimum effective size, which according to Meuwissen and Woolliams (994) may balance inbreeding depression, and it approaches the value of the estimated minimum viable effective size for endangered species conservation (Franklin, 980; Lande and Barrowclough, 987). The inbreeding rate of greater than % indicates that the current MF population should be considered endangered, but the discrepancy from the recommended value is not alarming (+0.09%). Thus, the demographic and genetic aspects of the population suggest that, although the breed is endangered, careful genetic management of the breed may help minimize inbreeding practices and enhance genetic variation. To this end, measures need to be put in place to contain the inbreeding rate, increase the effective population size, and as already outlined, promote the practice of selecting individuals for mating when relationships are less than 2.5%. The major hurdle to a widespread implementation of this practice is the cost of transporting jennies to off-farm stallion services. The use of AI and other new breeding technologies (Flores et al., 2008; Miró et al., 2009), and thus the potential for a jack to breed mares far away from its station, might be able to enhance the extent and results of such practices. Moreover, most jennies have ovulatory estrus throughout the year and cooled semen could also be used during the short-day-length season (Contri et al., 200). However, careful genetic management of the breed is an expensive process that cannot be paid for entirely by breeders, especially considering the underlying global economic crisis. Fortunately, a better future for the MF donkey is possible. The greater awareness and sensitivity of the Apulian Regional Administration regarding the need to safeguard local domestic animal diversity resulted in the approval of a comprehensive regional law on May 3, 2009, which provided for the protection and conservation-oriented use of native Apulian agricultural and Figure 2. Dispersal of effective population size of each replicate obtained on the basis of the relationship coefficient of matings.

forest resources, encompassing natural and cultivated plant species as well as farm animals, including the MF donkey. LITERATURE CITED Boichard, D., L. Maignel, and É. Verrier. 997. The value of using probabilities of gene origin to measure genetic variability in a population. Genet. Sel. Evol. 29:5 23. Bramante, G., and E. Pieragostini. 2005. Apulian native breeds: Present and future. Proc. 3th Congr. Fe.Me.S.P.Rum, Bari, Italy. Accessed Feb. 4, 20. http://www.kassiopeagroup.com/ it/pdf/xiii_femesprum_abstract.pdf. Caballero, A., and M. A. Toro. 2000. Interrelations between effective population size and other pedigree tools for the managements of conserved populations. Genet. Res. Camb. 75:33 343. Carluccio, A., S. Panzani, U. Tosi, P. Riccaboni, A. Contri, and M. C. Veronesi. 2008. Morphological features of the placenta at term in the Martina Franca donkey. Theriogenology 69:98 924. Cassell, B. G., V. Adamec, and R. E. Pearson. 2003. Effect of incomplete pedigree on estimates of inbreeding and inbreeding depression for days to first service and summit milk yield in Holsteins and Jerseys. J. Dairy Sci. 86:2967 2976. CBD. 992. Convention on Biological Diversity. Accessed Feb. 4, 20. http://www.cbd.int/doc/legal/cbd-en.pdf. Cecchi, F., R. Ciampolini, E. Ciani, B. Matteoli, E. Mazzanti, M. Tancredi, and S. Presciuttini. 2006. Demographic genetics of the endangered Amiata donkey breed. Ital. J. Anim. Sci. 5:387 39. Contri, A., I. De Amicis, M. C. Veronesi, M. Faustini, D. Robbe, and A. Carluccio. 200. Efficiency of different extenders on cooled semen collected during long and short day length seasons in Martina Franca donkey. Anim. Reprod. Sci. 20:36 4. Flores, E., E. Taberner, M. M. Rivera, A. Peña, T. Rigau, J. Miró, and J. E. Rodríguez-Gil. 2008. Effects of freezing/thawing on motile sperm subpopulations of boar and donkey ejaculates. Theriogenology 70:936 945. Folch, P., and J. Jordana. 998. Demographic characterization, inbreeding and maintenance of genetic diversity in the endangered Catalonian donkey breed. Genet. Sel. Evol. 30:95 20. Franklin, I. R. 980. Evolutionary change in small populations. Pages 35 in Conservation Biology: An Evolutionary-Ecological Perspective. M. E. Soule and B. A. Wilcox, ed. Sunderland, MA. Gandini, G., and P. De Filippi. 998. Minbreed Software package for the genetic management of small breeds. Proc. 6th World Congr. Genet. Appl. Livest. Prod., Armidale, Australia 27:45. Gandini, G., L. Ollivier, B. Danell, O. Distl, A. Georgoudis, E. Groeneveld, E. Martyniuk, J. A. M. van Arendonk, and J. A. Woolliams. 2004. Criteria to assess the degree of endangerment of livestock breeds in Europe. Livest. Prod. Sci. 9:73 82. Gandini, G., and E. Villa. 2003. Analysis of the cultural value of local livestock breeds: A methodology. J. Anim. Breed. Genet. 20:. Genetic diversity in the Martina Franca donkey 3 Gutiérrez, J. P., I. Cervantes, and F. Goyache. 2009. Improving the estimation of realized effective population sizes in farm animals. J. Anim. Breed. Genet. 26:327 332. Gutiérrez, J. P., I. Cervantes, A. Molina, M. Valera, and F. Goyache. 2008. Individual increase in inbreeding allows estimating effective sizes from pedigrees. Genet. Sel. Evol. 40:359 378. Gutiérrez, J. P., and F. Goyache. 2005. A note on ENDOG: A computer program for analysing pedigree information. J. Anim. Breed. Genet. 22:72 76. Gutiérrez, J. P., J. Marmi, F. Goyache, and J. Jordana. 2005. Pedigree information reveals moderate to high levels of inbreeding and a weak population structure in the endangered Catalonian donkey breed. J. Anim. Breed. Genet. 22:378 386. Kugler, W., H. P. Grunenfelder, and E. Broxham. 2008. Donkey Breeds in Europe. Inventory, Description, Need for Action, Conservation. Report 2007/2008. Monitoring Institute for Rare Breeds and Seeds in Europe/SAVE foundation, St. Gallen, Switzerland. Accessed Feb. 4, 20. http://www.boerenvee.nl/ uploads/ezelstudie.pdf. Lacy, R. C. 989. Analysis of founder representation in pedigrees: Founder equivalents and founder genome equivalents. Zoo Biol. 8: 23. Lande, R., and G. F. Barrowclough. 987. Effective population size, genetic variation, and their use in population management. Page 87 in Viable Populations for Conservation. M. E. Soule, ed. Cambridge University Press, Cambridge, UK. Lutaaya, E., I. Misztal, J. K. Bertrand, and J. W. Marbry. 999. Inbreeding in population with incomplete pedigrees. J. Anim. Breed. Genet. 6:475 480. Meuwissen, T. H. E. 999. Operation of Conservation Schemes. Page 9 in Genebanks and the conservation of farm animal genetic resources. J. K. Oldenbroek, ed. DLO Institute for Animal Science and Health, Lelystad, the Netherlands. Meuwissen, T. H. E., and Z. Luo. 992. Computing inbreeding coefficients in large populations. Genet. Sel. Evol. 24:305 33. Meuwissen, T. H. E., and J. A. Woolliams. 994. Effective sizes of livestock populations to prevent a decline in fitness. Theor. Appl. Genet. 89:09 026. Miró, J., E. Taberner, M. Rivera, A. Peña, A. Medrano, T. Rigau, and A. Peñalba. 2009. Effects of dilution and centrifugation on the survival of spermatozoa and the structure of motile sperm cell subpopulations in refrigerated Catalonian donkey semen. Theriogenology 72:07 022. Oldenbroek, J. K. 999. Introduction. Page in Genebanks and the Conservation of Farm Animal Genetic Resources. J. K. Oldenbroek, ed. DLO Institute for Animal Science and Health, Lelystad, the Netherlands. Van Doormaal, B. J., F. Miglior, G. J. Kistemaker, and P. Brand. 2005. Genetic diversification of the Holstein breed in Canada and internationally. Interbull. Bulletin No. 33. Accessed Feb. 3, 20. http://www-interbull.slu.se/bulletins/bulletin33/ Van%20Doormal.pdf.