Untersuchung zur Optimierung des Embryotransfers beim Pferd
Transcription
Untersuchung zur Optimierung des Embryotransfers beim Pferd
Aus dem Institut für Tierzucht und Tierhaltung der Agrar- und Ernährungswissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel Untersuchung zur Optimierung des Embryotransfers beim Pferd unter Feldbedingungen für die Nutzung im Rahmen von Zuchtprogrammen Dissertation zur Erlangung des Doktorgrades der Agrar- und Ernährungswissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von M.Sc. agr. Henrik von der Ahe aus Münster, Nordrhein-Westfalen Dekanin: Prof. Dr. K. Schwarz 1. Berichterstatter: Prof. Dr. G. Thaller 2. Berichterstatter: Prof. Dr. E. Schallenberger Tag der mündlichen Prüfung: 10. Mai 2012 Die Dissertation wurde dankenswerterweise finanziell durch die H. Wilhelm Schaumann Stiftung unterstützt. Table of contents General Introduction …………………………………………………………………………………………………1 Chapter One Analysis of genetic and management factors affecting ovulation and embryo recovery rate in German Warmblood donor mares …………………………………………………………………………………………………7 Chapter Two Impact of reproductive history and semen parameters on the number of recovered embryos in German Warmblood donor mares ………………………………………………………………………………………………..29 Chapter Three Effects on success of pregnancy rate after embryo transfer in warmblood recipient mares and comparison of embryo transfer foals to non – embryo transfer foals ………………………………………………………………………………………………..47 General Discussion ………………………………………………………………………………………………..71 General Summary ………………………………………………………………………………………………..87 Zusammenfassung …………………………………………………………………………………………..……91 General Introduction The use of equine embryo transfer has increased steadily since its first attempts in the early 1970s. Today it is commonly used in equine practice. Causes why embryo transfer is performed are various. It allows the production of offspring from show mares that compete in performance tests, like dressage or jumping, without being removed from their primary use. Increase of the annual reproduction rate in mares to more than one foal per year is possible. Not fully grown mares (2 years old) can be early used as donor mares for producing offspring through embryo transfer leading to a reduction of the generation interval, where otherwise insemination and following pregnancy of a two year old mare might not be performed. Subfertile mares, particularly those that suffer early embryo loss, can be used for producing offspring. The USA (15 000 transfers per year) and South America, especially Brazil (12 000 transfers per year), are leading in performing equine embryo transfer (Thibier, 2007). The use of equine embryo transfer in Europe is actually limited but numbers of recovered embryos and performed transfers are steadily increasing in the last decade. Important for the practitioner when performing ET are adapted monitoring and care of the donor mare, synchronization of donor and recipient and conducting embryo recovery (flushing) and following transfer itself (Hinrichs et al., 2005). Furthermore selection and management of the recipient mare plays an important role for achieving acceptable pregnancy rates after performing embryo transfer. There is no difference in monitoring and inseminating the donor mare, compared with the technique for a standard pregnancy. During estrous, follicular development on the 1 ovaries and changes in the uterus are monitored daily with ultrasonography assuring that the number of follicles ovulated and their exact day of ovulation, are known. Embryo recovery rate in young, fertile mares lies in a range between 55 to 80% (Hinrichs, 1993). The efficiency of equine embryo transfer would greatly increase, if it would be possible to superovulate donor mares. However, superovulation of donor mares, like in cattle, inducing multiple ovulation is ineffective in horses, because of various respond to FSH products of the donor mare. This lack of consistence superovulatory regimen in the donor leads generally to a limit of single embyo recovery per cycle. Although there is a breed dependent occurrence of multiple ovulation, especially in Quarter Horse and warmblood mares, guiding to higher embryo recovery rates and therefore a greater success of embryo transfer. Insemination of donor mares is generally realized with fresh/cooled or frozen-thawed semen 1 to 2 days prior to ovulation. Flushing of the uterus for recovering the embryos is generally done on day 7 or 8, unless the embryo is used for freezing. In this case flushing will be done on day 6, because between day 6 and 7, a acellular capsule is formed underneath the zona pellucida which may avoid the complete penetration of the cryoprotectant into the embryo (Seidel et al., 1986). The earliest successful recovery of the embryo is possible 144 to 156 h after ovulation because of the long transport, in comparison to other species, in the fallopian tube (Battut et al., 1997; Battut et al., 2001). At day 9 the embryo has grown to a size where danger increases to damage the embryo due to recovery, storing and transfer (McKinnon et al., 1988; Carnevale et al., 2000). There are several factors influencing the embryo recovery rate. Main sections are the day of recovery (Griffin et al., 1981; Squires et al., 1995), number of ovulations (Squires et al., 1987; Squires et al., 1987), age of the donor (Squires et al., 1982; 2 Vogelsang et Vogelsang, 1989) as well as the quality of sire’s semen (Francl et al., 1987). Factors that influence the pregnancy rate after transfer include the synchrony of donor and recipient mare, management of the recipient, method of transfer as well as the quality of the embryo. Highest pregnancy rates were obtained, if the recipient mare ovulates 1 d before to 3 d after the donor (McKinnon et al., 1988; Squires et al., 1995). The recipient mare should be examined with ultrasonography at day 4 and 5 after ovulation, if a corpus luteum has generated and no fluid or air was in the uterus (Squires et al., 1999). Method of transfer can be performed as surgical flank incision or as nonsurgical transfer, through the vagina of the recipient mare. Surgical transfer promised a consistent pregnancy rate of 65 to 75% (Squires et al., 1995), whereas results of nonsurgical transfer vary among technicians, with obtained pregnancy rates of 50 to 75% (Hinrichs, 1993, McKinnon et al., 1988; Riera et al., 1993; Squires et al., 1995; Vogelsang et al., 1985). As a result of its simplicity and in terms of animal welfare, the nonsurgical transfer is actually the most common technique. Size of the recipient mare in comparison to donor mare and service stallion is an aspect which is often discussed. Transfer into smaller recipients resulted in a retardation of fetus growth, which could not totally compensated (Allen et al., 2004). In general, the transfer of warmblood embryos into trotter or thoroughbred mares, which had commonly a similar size can sometimes result in a slightly reduced size at birth, but this is fully compensated during postnatal development. No investigations regarding differences in embryo transfer foals versus non embryo transfer foals have done to the best of our knowledge so far. However there are some studies about length of gestation, withers height at birth and weight at birth (Valera et al. 2006, Cilek 2009, Satuè 2011). 3 In Chapter 1 of the present study, fixed and random effects regarding the embryo recovery rate were investigated using a threshold mixed model and a probit link function. Age of the donor mare at time of flushing, season of flushing, preparation of the donor mare and the running number of flushing within season were investigated. Estimation of heritabilities for the traits “ovulation rate” and “number of recovered embryos” as well as phenotypic and genetic correlations between maternal and paternal components were performed. Additional research was conducted in Chapter 2 to investigate the breeding status of the donor mare as well as the quality of the semen. Finally in Chapter 3 the pregnancy rate of the recipient mare was investigated with respect to the age of the recipient mare at time of transfer, season (month of transfer), preparation of the recipient mare before transfer, synchrony between donor and recipient mare and the age of the embryo at time of transfer. The comparison of embryo transfer foals and non embryo transfer foals as well as colts and fillies was done with a view to the traits length of gestation, withers height at birth and weight at birth. REFERENCES Allen, W. R., Wilsher, S., Tiplady, C. and Butterfield, R. M.; The influence of maternal size on pre- and postnatal growth in the horse: III Postnatal Growth. Reproduction 2004; 127: 67-77 Batut, I., Colchen, S., Fieni, F., Tainturier, D., Bruyas, JF. Success rates when attempting to nonsurgically collect equine embryos at 144, 156 or 168 hours after ovulation. Equine Vet J (Suppl) 1997;25:60-2 Batut, I., Grandchamp des Raux, A., Nicaise, JL, Fieni, F., Tainturier, D., Bruyas, JF. When do equine embryos enter the uterine cavity? An attempt to answer. R&W Publications, Newmarket; 2001 Carnevale, EM, Ramirez, RJ, Squires, EL, Alvarenga, MA, Vanderwall, DK, McCue, PM. Factors affecting pregnancy rates and early embryonic death after equine 4 embryo transfer. Theriogenology 2000; 54; 965-79. Francl, A. T.,R. P. Amann,E. L. Squiresand B. W. Pickett. Motility and fertility of equine spermatozoa in a milk extender after 12 hours or 24 hours at 20degrees-C. Theriogenology 1987;27:517-525. Griffin, J. L.,R. S. Castleberryand H. S. Schneider. Influence of day of collection on recovery rate in mature cycling mares. Theriogenology 1981;15:106-106 Hinrichs, K. Embryo-Transfer in the mare - A status-report. Animal Reproduction Science 1993;33:227-240 Hinrichs, K.and Y.-H. Choi. Assisted Reproductive Techniques in the Horse. Clinical Techniques in Equine Practice 2005;4:210-218 McKinnon, EL. Morphologic assessment of the equine embryo. J Amvetmed Ass 1988;192:401-6 Riera, FL., and McDonough, J.; Commercial embryo transfer in polo ponies in Argentina. Equine Vet. J. 1993; 15(Suppl): 116-119. Seidel, GE Jr.; Cryopreservation of equine embryos. In Squires EL (ed), Veterinary Clinics of North America, Equine Practice: Diagnostic Techniques and Assisted Reproductive Technology 1986;12(1):85-101. Squires, E. L.,K. J. Imel,M. F. Iulianoand R. K. Shideler. Factors affecting reproductive efficiency in an equine embryo transfer programme. J Reprod Fertil Suppl 1982;32:409-414 Squires, E. L.,M. G. McClain,O. J. Gintherand A. O. McKinnon. Spontaneous multiple ovulation in the mare and its effect on the incidence of twin embryo collections. Theriogenology 1987;28:609-613. Squires, E. L.,A. O. McKinnon,E. M. Carnevale,R. Morrisand T. M. Nett. Reproductive characteristics of spontaneous single and double ovulating mares and superovulated mares. J Reprod Fertil Suppl 1987;35:399-403. Squires, E.and G. Seidel. Collection and transfer of equine embryos. Animal Reproduction and Biotechnology Bulletin No 11. Fort Collins CO: Colorado State University 1995:7-9 11-15 27-32 Squires, E. L., Mc Cue, P.M. and Vanderwall, D. The current status of equine embryo transfer. Theriogenology 1999;51:91-104. Thibier, M.; New records in the number of both in vivo-derived and in vitro-produced bovine embryos around the world in 2006. IETS Newsletter 2007;25(4):15-20. 5 Valera, M., Blesa, F., Dos Santos, R. and Molina, A.; Genetic study of gestation length in andalusian and arabian mares. Animal Reproduction Science 2006; Vol. 95, Issue 1-2: 75-96. Vogelsang SG, Bondioli, KR and Massey, JM.; Commercial application of equine embryo transfer. Equine Vet. J. 1985; (3): 89-91. Vogelsang, S. G.and M. M. Vogelsang. Influence of donor parity and age on the success of commercial equine embryo transfer. Equine Veterinary Journal 1989;21:71-72. 6 Chapter One: Analysis of genetic and management factors affecting ovulation and embryo recovery rate in German Warmblood donor mares H. von der Ahea, J. Tetensa, E. Stamerb, E. Kalma and G. Thallera a b CAU, Institute of Animal Breeding and Husbandry, Hermann-Rodewald-Straße 6, 24098 Kiel, Germany TiDa GmbH, Bosseer Str. 4c, 24259 Westensee/Brux, Germany Submitted for publication in Theriogenology 7 Abstract Embryo transfer (ET) is an important and frequently used biotechnology in animal breeding. A main motivation to apply ET in horses is the possibility to obtain foals from mares that are competing as sport horses. The success of ET is to a large extent determined by the number of transferable embryos obtained from the donor mare. Selection, management and flushing of the donor as well as paternal fertility are factors influencing embryo recovery. The primary aims of this study were thus to determine influencing factors including the donor mare’s age and management. Furthermore, heritabilities as well as phenotypic and genetic correlations for the traits “ovulation rate” (OR) and “number of recovered embryos” (RE) were estimated. Data for these traits were obtained from 5 715 flushes of 715 donor mares from a single stud. Threshold mixed models using a probit link function were applied. The OR was found to be significantly affected by the age of the donor mare and the season as well as the number of flushings within season and the preparation of the donor mare. The fixed effects of age, season and preparation of the donor mare, but not the number of flushings showed significant influences on the trait RE. As to the preparation, the application of an ovulation inducing agent had the highest effect, probably due to the optimized insemination time. The estimated maternal heritability of OR was low. For RE, the paternal genetic component has to be considered additionally and the estimates for both genetic components were also low. Notably, the paternal component seems to have a stronger impact on RE. The correlation of OR and RE was slightly positive whereas genetic correlations of maternal and paternal components are negative indicating a genetic antagonism. In summary, several management factors significantly affecting OR and RE were identified and genetic components were demonstrated. Although low, these components offer a way to genetically improve the respective fertility traits. 8 Keywords: horse, ET, German Warmblood, ovulation rate, embryo recovery, heritability Introduction Today, ET is commonly used in some countries in equine reproduction practice, both for the production of more offspring from a given mare and to obtain foals from mares that suffer early embryo loss [1]. Furthermore, ET is used to produce offspring from mares competing in any discipline at performance events, which naturally restricts their ability to deliver foals. For economic reasons, the genetic improvement of reproductive traits including the outcome of ET is of great importance for horse breeders. On the other hand, the breeding goals for (sport-) horses are mainly focused on jumping or dressage abilities. Success of ET is influenced by several factors such as the ovulation rate, the day of recovery, the age of the donor mare, and the quality of the semen. Monitoring and inseminating the donor mare does principally not differ from the technique of standard artificial insemination. During estrus, follicular development and uterine status are monitored daily with ultrasonography to assess the number of follicles and their exact time of ovulation. Donor mares are inseminated with fresh/cooled or frozen-thawed semen 1 to 2 days prior to ovulation. After successful fertilization, the developing embryo moves from the oviducts into the uterus at day 5 after ovulation and continues to grow rapidly in diameter. The donor mare’s uterus is typically flushed at day 7 or 8 to, unless the embryos are to be frozen. In such cases the recovery is performed at day 6, because at day 7 or 8 embryos have formed a capsule which leads to a lower pregnancy rate [2] due to an incomplete permeation of the freezant and resulting difficulties during the freezing/thawing process. Embryos recovered at day 8 after ovulation are larger and easier to locate in the filter or Petri 9 dish [1]. Thus, some embryos might be missed if the flush is performed at an earlier stage [1]. A day 9 embryo, however, has grown to a size, where pregnancy rates after transfer may be reduced due to potential damages to the embryo during handling. The influence of the day of flushing on the number of recovered embryos (RE) has been considered in several studies. Griffin et al. [3] used sixteen mature mares and collected 24 embryos from 38 flushes five to nine days post ovulation. They reported the highest recovery rate of 80% (8 embryos / 10 flushes) for day 6 followed by day 8 and day 7 with 67% (4/6) and 60% (9/15), respectively. Other authors did not find significant differences between days 7-10 [4]. Horses are generally a monovular species, but there are reports that multiple ovulations occur with frequencies ranging from 4 to 43 % [5,6]. Several aspects such as age, breed, genetics, season, nutrition and reproductive status were investigated in these studies and found to substantially influence findings of multiple ovulations in the mare [5-8]. The frequency of multiple ovulations in draft horses and Thoroughbreds ranges from 15 to 30 %, whereas light breeds and ponies showed a lower rate of around 2 to 10 % [5,6]. Breed differences as well as individual differences between mares indicate a genetic background for the occurrence of multiple ovulations. The recovery of two or more embryos from a multiple ovulating mare can substantially increase the reproductive efficiency of an embryo transfer program [7]. The chances for obtaining a pregnancy from a given donor mare may be doubled if both embryos are viable. For embryos collected from mares seven days after ovulation, Squires et al. [9] reported recovery rates of 53% for single-ovulating mares compared to 106% for double-ovulating mares. In cattle, it is common practice to induce a superovulation in the donor in order to obtain a higher number of embryos. In contrast, the majority of donors in the horse are spontaneous, singleovulating mares [10]. Recent studies indicate an effective increase in the number of 10 ovulations and embryos recovered in mares treated with recombinant equine FSH and LH [11]. However, the success of superovulation schemes in the horse is still limited and further research is needed to optimize dosage and regime [11]. Regarding the age of the donor mare, Vogelsang and Vogelsang [12] reported significant differences between embryo recovery rates in younger compared with older mares. Mares younger than 9 years showed a recovery rate of 60.6%, while mares older than 17 years had a rate of only 30.1%. The success of ET can be regarded as a complex trait influenced by a variety of environmental and genetic factors including direct, maternal and paternal components. The aim of the current study was to quantify potential determinants affecting the success of embryo recovery. The analyses are focused on factors related to the donor mare’s management including the age and treatment of the donor mare and the season as well as the number of flushings within a season and the day of flushing. Furthermore, heritabilities and genetic correlations were estimated to provide parameters necessary to improve equine embryo transfer within the context of breeding programs. Interactions between paternal and maternal genetic components were analyzed to facilitate a differentiated insight into the ET status of horses. To the best of our knowledge, this study is the first to examine genetic aspects of ET in horses. Material and Methods The data set comprised reproduction records related to embryo transfer of 715 Warmblood donor mares obtained from a large stud farm in Northern Germany during the years 2004 to 2008. Data on ovary and uterus control as well as on inseminations and flushes were registered daily by veterinarians. ET donor mares were monitored and inseminated in the same manner as mares used in the standard 11 breeding program. Mares were generally inseminated either with fresh or frozen semen, one to two days prior to ovulation. Embryos were obtained six to nine days after ovulation by flushing the uterus via cervix. Data were immediately recorded manually and transferred to electronic media later on. ET reproduction traits considered were the ovulation rate (OR) and the number of recovered embryos (RE). The data comprised a total of 5 755 cycles of donor mares. In 40 cases, the donor mare did not ovulate, so that flushing was not possible. Furthermore, several incomplete records had to be removed resulting in 5 665 and 5 674 flushes for the analysis of OR and RE, respectively. Due to the comparatively small number of ovulations with more than two ova OR was defined as a binary trait and coded with ‘0’ for one ovum and ‘1’ for at least two ova. RE was also coded as a binary trait with no flushed embryo (‘0’) vs. at least one embryo (‘1’). An overview is given in Table 1. Table 1 Overview of the analyzed reproduction traits ovulation rate (OR) and recovered embryos per flush (RE) Trait Analyzed Frequency by trait class (N) Mean flushes (N ) Ovulation rate 5 665 (OR) Embryos recovered (RE) 5 674 1 ovum ≥ 2 ova 4 424 1 241 (78.1%) (21.9%) no embryo ≥ 1 embryo 2 909 2 765 (51.3%) (48.7%) 1.22 ova/flush 0.54 RE/flush The 2 765 positive flushes resulted in a total of 3 050 recovered embryos with one (2 506), two (252), three (15) and four (1) embryos per flush. 12 The donor age (‘Age’) at flushing was grouped into eight classes as 2, 3, 4, 5, 6 to 8, 9 to 11, 12 to 15 and ≥ 16 years old. The season of flushing (‘Season’) classes correspond to month’s classes within years. Donors were flushed routinely between March and September. The average number of performed flushes per month was 159. Treatment code (‘TC’) represents six different combinations of time of treatment before insemination (last and penultimate treatment) and type of treatment (heat and ovulation inducing agents, Table 2). The penultimate treatment was only considered if it occurred within a time range of less than 28 days before flushing. Table 2 Definitions and distribution of treatment codes (TC). TC 3 was excluded from further analyses due to a small number of observations last treatment penultimate treatment < 28 d TC N % before flushing none none 0 352 6.17 heat inducing agent none or >28 d 1 169 2.96 heat inducing agent heat inducing agent 2 106 1.86 heat inducing agent ovulation inducing agent 3 30 0.53 ovulation inducing agent none or >28 d 4 2999 52.54 ovulation inducing agent heat inducing agent 5 1804 31.60 ovulation inducing agent ovulation inducing agent 6 248 4.34 Number of flushing (‘NOF’) within breeding season was in a range between 1 and 15 and was divided into nine classes (1, 2, 3, 4, 5, 6, 7, 8, ≥9). The ‘day of flushing’ (‘Day’) was defined as the difference between the date of flushing and the date of ovulation and ranged from day 6 to day 10. Most of the flushes were performed at days 7 and 8 (74 %). 13 Data analysis was done applying a threshold mixed model and a probit link function within the GLIMMIX procedure of the SAS package (SAS 2008). The following model was used to analyse the ovulation rate (OR): Φ , where is the expected probability to observe at least two ovulations and Φ is the cumulative probability function of the standard normal distribution. , , and represent the fixed effects of i-th age class (i = 1,..., 8), the j-th month of flushing (j = 1,..., 36), the k-th preparation of the donor (k = 1,..., 6) and the l-th flushing number within season (l = 1,..., 9), respectively. The random effect of the m-th donor mare is represented by (m = 1,..., 715). The recovery rate was analysed using the following model: Φ !" #$$% , where is the expected probability to obtain at least one embryo and Φ is the cumulative probability function of the standard normal distribution. , , , and !" represent the fixed effects of i-th age class (i = 1,..., 8), the j-th month of flushing (j = 1,..., 36), the k-th preparation of the donor (k = 1,..., 6), the l-th flushing number within season (l = 1,..., 9), and the m-th day of flushing after ovulation (l= 1,...,5), respectively. The random effects of the n-th donor mare and the o-th service stallion are represented by (n = 1,..., 715) and #$$% (o = 1,..., 182), respectively. 14 To estimate the heritabilities for OR and RE as well as phenotypic and genetic correlations between traits as well as maternal and paternal components, random effects of mare and stallion were divided into the permanent environmental effect and the additive genetic effect of the animal based on an additive genetic relationship matrix. The available pedigrees contained four generations and comprised 5 092 animals in total. The number of sires was 188 with 3.8 daughters on average. For estimation of variance components and correlations the software package ASReml 3.0 [13] was used. Results Within the time period from 2004 to 2008, the average number of recovered embryos per flushing was 0.54 across 715 analyzed donor mares. The average number of ovulations per flush was 1.22 ± 0.50, the probability of obtaining at least two ovulations was 21.9 %. All analyzed fixed effects (‘Age’, ‘Season’, ‘TC’, ‘NOF’) have a highly significant effect on OR. With an increasing age of the donor mare from 2 to 15 years, the probability of more than one ovulation increased from 0.13 to 0.30, respectively (Figure 1). Only donor mares belonging to the age group ≥16 years showed a lower probability for multiple ovulations (0.23). With a view to the ‘Season’ the highest probability for at least two ovulations was in April 2004 (0.29), the lowest probability in July 2008 (0.12, Figure 2). Regarding the treatment code (‘TC’), code 5 indicates the highest probability for multiple ovulations (0.27, Figure 3). The lowest probability for multiple ovulations was obtained by code 0 and 1 (both 0.14). The number of flushes within season (‘NOF’) showed that with increasing number of flushes the probability for multiple ovulations also increased (class 1: 0.12 to class 9: 0.28, Figure 4). 15 Fixed effects of ‘Age’, ‘Season’, and ‘TC’ – but not the flushing number within breeding season (‘NOF’) and the day of recovery (‘Day’) – had a significant influence on the trait RE. Within the donor’s age range of 2 to 15 years, minor differences were detected in the likelihood of recovering an embryo (0.41 to 0.46). For mares older than 15 years, the probability of recovering an embryo decreases to 0.30 (Figure 1) leading to a significant difference in the number of provided embryos between donors older than 15 and younger than 16 years. Figure 1 Probability of >1 ovulation and to recover at least 1 embryo conditional on the age of the donor mare (F-test:0.0001; LS-Means). Different letters indicate significant differences in least square means (p<0.05). Season of flushing affected the probability of recovering an embryo in a range of 0.22 to 0.60. The recovery rate was higher in spring and early summer than in late summer and autumn (Figure 2). 16 Figure 2 Probability of >1 ovulation (upper figure) and to recover at least 1 embryo (lower figure) conditional on season (month class within breeding season). Given are the LS-Means together with the respective standard errors (error bars). The probability to obtain at least one embryo was significantly higher in mares whose last treatment was an ovulation inducing agent compared to those having received a heat inducing agent as their last treatment or no treatment at all. This seems more or less independent from the penultimate treatment (Figure 3). The combination of heat and ovulation inducing agent (‘TC’ = 3) was, however, not tested due to a small number of observations. Notably, the probability in mares having received no treatment at all is significantly higher than in mares having received an heat inducing agent as last treatment and no ovulation inducing agent within 28 days prior to the flushing. 17 Figure 3. Probability of >1 ovulations and to recover at least 1 embryo depending on the treatment of the donor mare (F-test:0.0001; LS-Means). Different letters indicate significant differences in least square means (p<0.05), error bars indicate the respective standard error. Animals with treatment class ‘0’ (light grey bar) have received no treatment at all. White bars represent the application of an heat inducing agent (‘TC’ = 1 or 2) and grey bars the application of an ovulation inducing agent (‘TC’ = 4,5 or 6) as last treatment, respectively. The probability for a successful flushing conditional on the consecutive number of the flushing within breeding season is in a range of 0.38 (flush number 1) to 0.44 (flush number 6) and shows no significant influence on the embryo recovery rate (Figure 4). With respect to the day of flushing, the probability to recover at least one embryo ranged from 0.39 (Day 6) to 0.43 (Day 8) but shows no significant differences. 18 Figure 4 Probability of >1 ovulations and to recover at least 1 embryo conditional on the consecutive number of flushing within breeding season (F-test:0.0001; LSMeans), error bars indicate the respective standard error. Different letters indicate significant differences in least square means (p<0.05), error bars indicate the respective standard error. Animals with treatment class ‘0’ light grey bar) have received no treatment at all. White bars represent the application of an heat inducing agent (‘TC’ = 1 or 2) and grey bars the application of an ovulation inducing agent (‘TC’ = 4,5 or 6) as last treatment, respectively. The heritabilities for fertility parameters for RE (h2 = 0.037 ± 0.020) and OR (h2 = 0.144 ± 0.045) within donor mares were low (Table 3). The service stallion shows a slightly higher influence on RE than the donor mare (h2 = 0.045 ± 0.037). Table 3 Heritabilities and proportions of the effect of the permanent environment of mare and service stallion for RE and OR (standard errors in parentheses) Trait Animal permanent environment Heritability RE donor mare 0.030 ± 0.020 0.037 ± 0.020 RE service stallion 0.040 ± 0.034 0.045 ± 0.037 OR donor mare 0.099 ± 0.041 0.144 ± 0.045 donor The genetic correlation between donor mare and service stallion is -0.599 ± 0.484. The permanent environmental effect for RE is 0.030 ± 0.020 for the donor mare. The 19 service stallion exhibits a permanent environmental effect for the trait RE of 0.040 ± 0.034. For OR the permanent environmental effect of the donor mare is 0.099 ± 0.041. Correlation (and standard error) between the two traits RE and OR is 0.154 ± 0.283. Discussion In the period 2004 to 2008, 0.54 embryos per flush were achieved over all 715 donor mares. This value is in agreement with Douglas [14], Squires et al. [15]. and Vogelsang and Vogelsang [12] who found probabilities to flush an embryo ranging from 0.53 to 0.80 in young fertile mares. The average number of ova was 1.22 per cycle with a probability of 21.9% to observe more than one ovum. This is partly in agreement with previous reports. The frequency of multiple ovulations in draft horses and Thoroughbreds has been reported to range from 15 and 30 %, whereas that in light breeds and ponies was only up to 10% [5,6]. Based on these values, a slightly lower probability might have been expected in the German Warmblood horses analyzed within the current study. The breeding programs of the major German Warmblood associations have, at least in past decades, been coined by substantial introgression of Thoroughbreds. Many Warmbloods have high shares of Thoroughbred today of sometimes even more than 50%, which might partly explain the high probability of multiple ovulations in the analyzed data set. Another explanation might be an optimized preparation of the donors. While the probability for at least two ovulations was 21.9%, the probability to obtain at least two embryos was only 10% indicating a loss of 12% between the time of ovulation and flushing. Vanderwall [16] reported very high (>90%) pregnancy rates in young mares under optimal conditions (i.e., fresh, fertile semen) versus older mares that showed a lower pregnancy rate of 85%. Although high fertilization rates were 20 achieved, embryo recovery rates on days 6 to 9 after ovulation were distinctly lower for older mares [12,17], which indicates high embryonic losses during the first week of gestation. In other studies, the estimated embryonic loss rate between fertilization and day 14 was lower than 10% for young mares but up to 70% for older mares [18,19]. Vogelsang and Vogelsang [12] reported significant differences in embryo recovery rates in younger mares compared with older mares. While the recovery rate in mares younger than 9 years was more than 60%, it decreased to ~30% in mares older than 17 years. These reports are in agreement with the current study as an increasing probability for more than one ovulation was observed in old mares, while the embryo recovery rate decreased (Fig. 1). Thus there is an antagonism between OR and RE regarding the age of the donor mare. Causes of reduced recovery from older mares include uterine and oviductal pathology and an increasing early embryonic death [18] as well as defective oocytes and an impaired maturation [2022]. Several reports based on data from commercial operations have reported a 50 % embryo recovery rate from single-ovulating mares, some of which were subfertile mares [23,24]. In most breeds, a low recovery rate due to the lack of multiple ovulation increases the time required to obtain a pregnant recipient mare. This is associated with increased costs for the whole procedure of equine embryo transfer. Double or triple ovulations during a given cycle would result in higher embryo recovery rates than are currently possible from single-ovulating mares [9]. Therefore, superovulation would be desirable in mares and would highly increase the efficiency of equine embryo transfer. Superovulation in mares is, however, still difficult and generally embryos result only from the normally developing one (or sometimes two) dominant follicles per cycle. The main difficulty is substantial variation in the donor mare´s response to treatment. Although recent studies indicate the effectiveness of 21 recombinant equine FSH and LH [11], there is presently no product for superovulation which has gained widespread acceptance for clinical use in equine embryo transfer. Thus, most of the mares in an embryo transfer program are currently not stimulated. This also applies to the donor mares analyzed in the current study. However, the treatment with an ovulation inducing agent increased the probability to recover at least one embryo by approximately 0.05 (Figure 3). If the last treatment was an ovulation inducing agent, then ovulation rate is highest in those animals whose penultimate treatment was heat induction. However, the recovery rate after stimulated ovulation is high, almost irrespective of the penultimate treatment. Based on these results, the application of a heat inducing agent might be dispensable with respect to the number of recovered embryos. The mechanism by which the ovulation inducing agent increases the number of recovered embryos is most likely not related to a higher OR, but to an optimal time of insemination. The length of the mare’s estrus cycle ranges from 15 to 26 days the duration of estrus from 2 to12 days with a mean of 7 days [25]. Thus, the precise prediction of the time of ovulation depends on dense ultrasonsographic monitoring. The positive effect of stimulating the mare is the predictable time of ovarian maturation and following ovulation. By knowing the narrow time slot in which ovulation will occur, it is possible to inseminate the donor mare at the best time, which increases the number of recovered embryos. Especially when using cooled, frozen or poor-quality semen, insemination close to ovulation increases fertilization rates [26]. The number of ovulations as well as of recovered embryos is higher in spring and early summer than in late summer and autumn. It is known that the reproduction cycle of horses starts with an increasing day length. So the time of mating and giving birth in the horse is spring and early summer. The results for OR as well as for RE are in agreement with this. Hence, spring is the best time to start embryo recovery in 22 the year. Considering the consecutive number of the flushing within a season, no significant differences were found regarding the number of recovered embryos. This is not consistent with the result obtained from ovulation rate: the number of ovulations increases with increasing flushing number. One would thus expect a higher number of recovered embryos in later flushes. This discrepancy may be due to poorer quality of the ovulated oocytes. Generally, the flushing of donor mares took place from day 6 to day 9 after ovulation, which is a standard procedure [27]. Flushing at day 6 is, however, preferably performed if the embryo will be frozen after flushing. A reason for flushing the donor mare at day 8 or later is that some embryos, especially from older donors, may be missed if the flush is performed at an earlier stage [1]. In this study, no significant differences could be detected between the days of flushing. This is in agreement with a recent study by Jacob et al. [4], who reported recovery rates in a range 56 to 66% for days 7 to 10 with the highest value achieved for day 8 (66%, 285/434). The rates did not significantly differ between these days. The consistent performance in the current study might be due to the experienced veterinarians who performed the flushes. As to be expected for fertility parameters, the heritabilities for both analyzed traits are low, although the maternal heritability for OR is substantially higher than for RE (Table 3). Notably, the paternal heritability for the trait RE was higher than the maternal component indicating that the impact of paternal fertility on embryo recovery is higher than previously assumed. This likely applies to the pregnancy rate in standard artificial insemination programs as well. Thus, more attention should be given to paternal fertility traits in breeding programs. A major factor determining paternal fertility is the semen quality, which has been analyzed in an accompanying paper [28]. The correlation between RE and OR is slightly positive, whereas genetic 23 correlations between maternal and paternal components show a slightly negative tendency. Despite the comparatively large data set, the estimates are afflicted with high standard errors. Nevertheless, the results indicate a genetic antagonism, which is consistent with the situation found in cattle. Genetic correlations between maternal and paternal effects for most fertility traits are reported to be negative in this species [29]. König et al. [30] emphasized the need for studies revealing the physiological mechanisms responsible for the phenomenon of negative correlations between maternal and paternal effects for such traits. The current study is to our knowledge the first to estimate genetic parameters related to ET. Based on our results, we conclude that such studies should be carried out in the future not only for cattle but also for horses. The low heritability of the complex fertility traits limits the possibility to breed for an increased breeding performance. Nevertheless, the traits have a genetic component and the application of genomic selection in the horse, which will probably be implemented within the next years, might be a promising way to achieve genetic improvement of fertility. However, this substantially depends on the availability of accurate phenotypic information as used within the current study. Conclusion Within the current study, the use of young donor mares, the start of flushing in spring and an induced ovulation were found to increase the number of recovered embryos offering possibilities to optimize ET. The heritabilities for OR and RE were low as to be expected for fertility traits. Nevertheless, the genetic component offers a way to breed for improved fertility applying advanced breeding methodologies like genomic selection. Especially the paternal fertility seems to affect embryo recovery. Further 24 studies presented in an accompanying paper have thus addressed the influence of semen quality on reproductive outcome. Acknowledgments The authors thank the H. Wilhelm Schaumann Stiftung, Hamburg, for the financial support of H. von der Ahe. References 1. Hinrichs K, Choi Y-H. Assisted Reproductive Techniques in the Horse. Clinical Techniques in Equine Practice 2005;4: 210-218. 2. Hinrichs K. Embryo-Transfer in the mare - A status-report. Animal Reproduction Science 1993;33: 227-240. 3. Griffin JL, Castleberry RS, Schneider HS. Influence of day of collection on recovery rate in mature cycling mares. Theriogenology 1981;15: 106-106. 4. Jacob JCF, Haag KT, Santos GO, Oliveira JP, Gastal MO, Gastal EL. Effect of embryo age and recipient asynchrony on pregnancy rates in a commercial equine embryo transfer program. Theriogenology 2012;77: 1159-1166. 5. Ginther O. Reproductive Biology of the Mare. Basic and Applied Aspects. 2nd ed Cross Plaisn, WI: Equiservice 1992. 6. Bruyas JF, Battut I, Fieni F, Tainturier D. Gemellar gestation in a mare: A major cause of abortion. Point Veterinaire 1997;28: 43-53. 7. Squires EL, McKinnon AO, Carnevale EM, Morris R, Nett TM. Reproductive characteristics of spontaneous single and double ovulating mares and superovulated mares. J Reprod Fertil Suppl 1987;35: 399-403. 8. Pascoe RR, Pascoe DR, Wilson MC. Influence of follicular status on twinning rate in mares. J Reprod Fertil Suppl 1987;35: 183-189. 25 9. Squires EL, McClain MG, Ginther OJ, McKinnon AO. Spontaneous multiple ovulation in the mare and its effect on the incidence of twin embryo collections. Theriogenology 1987;28: 609-613. 10. Squires EL. Superovulation in mares. Vet Clin North Am Equine Pract 2006;22: 819-830. 11. Meyers-Brown G, Bidstrup LA, Famula TR, Colgin M, Roser JF. Treatment with recombinant equine follicle stimulating hormone (reFSH) followed by recombinant equine luteinizing hormone (reLH) increases embryo recovery in superovulated mares. Anim Reprod Sci 2011;128: 52-59. 12. Vogelsang SG, Vogelsang MM. Influence of donor parity and age on the success of commercial equine embryo transfer. Equine Veterinary Journal 1989;21: 71-72. 13. Gilmour AR, Gogel BJ, Cullis BR. ASReml User Guide, release 3.0. . 2009. 14. Douglas RH. Some aspects of equine embryo transfer. J Reprod Fertil Suppl 1982;32: 405-408. 15. Squires EL, Imel KJ, Iuliano MF, Shideler RK. Factors affecting reproductive efficiency in an equine embryo transfer programme. J Reprod Fertil Suppl 1982;32: 409-414. 16. Vanderwall DK. Early Embryonic Loss in the Mare. Journal of Equine Veterinary Science 2008;28: 691-702. 17. Woods GL, Hillman RB, Schlafer DH. Recovery And Evaluation Of Embryos From Normal And Infertile Mares. Cornell Veterinarian 1986;76: 386-394. 18. Ball BA, Little TV, Weber JA, Woods GL. Survival of day-4 embryos from young, normal mares and aged, subfertile mares after transfer to normal recipient mares. Journal of Reproduction and Fertility 1989;85: 187-194. 26 19. Ball BA, Little TV, Hillman RB, Woods GL. Pregnancy rates at Days 2 and 14 and estimated embryonic loss rates prior to day 14 in normal and subfertile mares. Theriogenology 1986;26: 611-619. 20. Carnevale E, Ginther O. Defective oocytes as a cause of subfertility in old mares. Biol Reprod Monogr 1 (Equine Reproduction VI) 1995: 209-214. 21. Brinsko SP, Ball BA, Ellington JE. In-vitro maturation of equine oocytes obtained from different age-groups of sexually mature mares. Theriogenology 1995;44: 461-469. 22. Bézard J. In vitro fertilization in the mare. Proceedings of the International Scientific Conference on Biotechnics in Horse Reproduction 12 Crakow, Poland (Abstract) 1992. 23. Vogelsang SG, Bondioli KR, Massey JM. Commercial application of equine embryo transfer. Equine Veterinary Journal 1985;17: 89-91. 24. Squires E, Seidel G. Collection and transfer of equine embryos. Animal Reproduction and Biotechnology Bulletin No 11 Fort Collins CO: Colorado State University 1995: 7-32. 25. Senger PL. Pathways to Pregnancy and Parturition, 2nd edition: Current Conceptions Inc., 2011. 26. Samper JC, Jensen S, Sergeant J, Estrada A. Timing of induction of ovulation in mares treated with Ovuplant or Chorulon. Journal of Equine Veterinary Science 2002;22: 320-323. 27. Squires EL, McCue PM, Vanderwall D. The current status of equine embryo transfer. Theriogenology 1999;51: 91-104. 28. Von der Ahe H, Stamer E, Kalm E, Thaller G, Tetens J. Theriogenology 2013. 27 29. Pasman E, Reinhardt F. Beschreibung der Zuchtwertschätzverfahren für Fruchtbarkeit und Abkalbemerkmale im VIT bei Schwarzbunt, Rotbunt und Rotvieh. DGFZ Schriftenreihe 1998;11: 82-88. 30. König S, Bosselmann F, von Borstel UU, Simianer H. Genetic Analysis of Traits Affecting the Success of Embryo Transfer in Dairy Cattle. Journal of dairy science 2007;90: 3945-3954. 28 Chapter Two: Impact of reproductive history and semen parameters on the number of recovered embryos in German Warmblood donor mares H. von der Ahea, E. Stamerb, E. Kalma, G. Thallera and J. Tetensa a b CAU, Institute of Animal Breeding and Husbandry, Hermann-Rodewald-Straße 6, 24098 Kiel, Germany TiDa GmbH, Bosseer Str. 4c, 24259 Westensee/Brux, Germany Submitted for publication in Theriogenology 29 Abstract Success of embryo recovery in horses depends on several factors such as age and preparation of the donor mare, the day of flushing and the season. Furthermore, the reproductive status of the donor and the quality of the sire’s semen play an important role. The aim of the current study was to investigate effects of the donor mare’s breeding history on the ovulation rate (OR) and the number of recovered embryos (RE). Furthermore, the impacts on embryo recovery of semen type (fresh vs. frozen) and quality (number and motility of spermatozoa in fresh semen) were analyzed. The data set consisted of a total of 5 715 flushes from 715 donor mares sired by 188 service stallions. The breeding status of donors showed significant effects on the ovulation rate, but notably not on the embryo recovery rate. The type of semen and the motility of the spermatozoa also exhibited a significant impact on the embryo recovery rate. Regarding the semen type, fresh semen was clearly superior to frozen semen. In fresh semen, the proportion of motile spermatozoa substantially determines the success of embryo recovery. Keywords: horse, ET, German Warmblood, ovulation rate, embryo recovery, semen quality, reproductive history Introduction Equine embryo transfer (ET) has gained importance in horse breeding over the last ten years, predominantly to increase the number of foals from show mares or mares with successful offspring. In an accompanying study based on the same large data set, it has been shown, that the age and the preparation of the donor mare as well as the season affect the success flushing [1]. However, embryo recovery requires successful insemination and thus also depends on paternal fertility. The important role of the paternal genetic component was also demonstrated in the aforementioned 30 study [1]. Furthermore, Vidament et al. [2] reported that the breeding history and the management of the mare during the estrus period as well as the quality of the semen are the most important factors for achieving a pregnancy, especially when using frozen semen. The breeding history of the mare includes the age, the reproductive performance of previous breeding seasons as well as the breeding status, i.e. whether the mare was pregnant or barren in the last breeding season or is a maiden mare. The reproductive history is one of the main factors that have to be considered when selecting a mare as a donor mare for embryo transfer [3-7]. The number of embryos decreased with a poor reproductive history and higher ages of the donor mare. Reasons for lower numbers of recovered embryos of older mares may be oviductal and uterine pathology as well as early embryonic death [8]. Furthermore, the chance to obtain an embryo from an old donor mare is low because of defective oocytes which may lead to reduced fertility in these old mares [7]. With respect to embryo transfer, pregnancy rates around day 7 are of great interest, because these are the days on which the donor mare is generally flushed after insemination. When investigating groups of young maiden mares (<7 years old), older maiden mares (>8 years old), barren mares and mares with foal at foot, Samper [9] found that the mare’s status had a big effect on pregnancy rates. The average first cycle pregnancy rates within the second week of gestation were 68.8%, 37.2%, 56.2% and 59.3% for each group, respectively. Samper et al. [10] reported that the number of older maiden mares (>8 years) with a prolonged sport or show career used for breeding is steadily increasing. Especially when bred with frozen semen, these mares had lower pregnancy rates and were potential candidates for failure. 31 The type and the quality of semen also affect embryo recovery. The use of fresh semen results in a higher recovery rate as compared to frozen semen [9]. Sanchez et al. [11] described the characteristics of frozen semen as follows: Advantages of frozen semen are (1) continuous availability of the semen, (2) better timing of insemination, (3) lower costs of transportation, (4) decrease in the risk of transmitting venereal diseases, and (5) increase of the genetic pool. Disadvantages are (1) lower pregnancy rates from some stallions, (2) increased cost to mare owner, (3) increased costs for semen preparation and (4) risk of disease transmission. The fertility of frozen semen is influenced by several factors including semen quality, individual difference between stallions and the insemination dose. It is well known that there is a variation in semen freezability among service stallions and further between ejaculates from the same stallion. Pregnancy rates obtained with frozen semen in commercial programs ranged from 32 to 73% per cycle [12] and from 26% [13,14] to 66% [15] per season with most values varying from 30 to 55% for the seasonal success rate [7,16,17]. The aim of the current study was to determine the influence of the mare’s reproductive status on the ovulation rate and the number of recovered embryos in German Warmblood horses. Furthermore, the effect of type and quality of semen on the number of recovered embryos was analyzed. Material and Methods The available data consisted of information about preparation, insemination and flushing of 715 Warmblood donor mares on a large stud farm located in Northern Germany. Data of 5 715 flushes were collected in a five year period ranging from 2004 to 2008. Donor mares were selected by the head of the stud, based on genetic 32 merit and not on reproductive history. Reproduction data (e.g. ovary and uterus control, inseminations and flushes) were registered daily by veterinarians. Donor mares were checked and artificially inseminated using the same procedures as for a standard artificial insemination and generally inseminated with fresh or frozen semen, one to two days prior to ovulation. Embryos were obtained six to nine days after ovulation by flushing the uterus via cervix. Analyzed reproduction traits were the ovulation rate (OR) and the number of recovered embryos (RE). From the total data set comprising 5 715 flushes, all incomplete records were removed including 40 cases with no ovulation at all. This resulted in 5 665 and 5 674 available flushes for OR and RE, respectively. Due to the small number of ovulations with more than two ova (1 241 flushes) OR was defined as binary trait with one ovum (‘0’) vs. at least two ova (‘1’). The 2 765 positive flushes (3 050 recovered embryos) resulted in the recovery of 1 (N= 2 506), 2 (N = 252), 3 (N = 15) or 4 (N = 1) embryos. The fraction of flushings with more than one embryo was hence only 9.66% and RE was also coded as binary trait with no flushed embryo (‘0’) vs. at least one embryo (‘1’). The data set has previously been described in detail by von der Ahe et al. [18]. Factors possibly influencing OR and RE considered with specific attention within the current study were the reproductive status of donor in the end of the previous breeding season (‘Status’), the use of fresh vs. frozen semen (‘Semen’) and the semen quality reflected by the parameters ‘number of spermatozoa’ (‘Mil’) and the ‘motility of spermatozoa’ (‘Mot’). The latter two parameters were only available for fresh semen. Starting the data acquisition in 2004 as the first flushing season of donor mares, the breeding status was set to missing, because there was no information on prior use of the donor. ‘Status’ was divided into three classes (Table 1): mares that were 33 pregnant at the end of the preceding breeding season, mares that were barren and mares that were used as donor mares in the previous season. For mares entering the procedure during time of data acquisition, the reproductive history was usually unknown. These mares were thus excluded from further analyses. Table 1 Definitions and number of observations for different classes of the reproductive status of the donor mare for the analyzed traits OR and RE Number of flushes per trait (N) Reproductive status OR RE 1 932 1 932 (53.19%) (53.11%) 338 338 season (9.31%) (9.29%) 3: Mare was already used as donor in 1 362 1 368 (37.50%) (37.60%) 3 632 3 638 1: Mare was pregnant in last breeding season 2: Mare was barren in last breeding last breeding season Total For the classification of the effect ‘Semen’, fresh semen was contrasted to frozen semen and semen collected from one ejaculation to semen collected from different ejaculations. Due to a small number of observations (N = 10) for fresh semen collected from different ejaculations, this class was pooled with fresh semen from a single ejaculation resulting in three different classes as summarized in Table 2. Table 2 Summary of the three classes of semen status Semen status Observations (N) 1: Fresh semen 1 955 (60.75%) 2: Frozen semen from one ejaculation 1 012 (31.45 %) 3: Frozen semen from different ejaculations 251 (7.80%) Total 3 218 34 The semen quality parameters ‘Mil’ (= number of spermatozoa) and ‘Mot’ (= motility of spermatozoa) were divided into 3 classes according to the number of spermatozoa per insemination and the proportion of motile spermatozoa (Table 3). Table 3 Definitions and number of observations for the semen quality parameters Number of spermatozoa per Proportion insemination (‘Mil’) Class Definition1 1 ≤100 mio Inseminations (N) 718 Definition1 ≤45 % 120-200 mio 449 Total 1 ≥ 250 mio 332 Inseminations (N) 486 (32.42%) 50-60% (29.95%) 3 motile spermatozoa (‘Mot’) (47.90%) 2 of 574 (38.29%) ≥65 % 439 (22.15%) (29.29%) 1 499 1 499 Number and motility of spermatozoa were not recorded on a continuous scale Further fixed effects included in the models were the age of the donor mare (‘Age’) with classes 2, 3, 4, 5, 6 to 8, 9 to 11, 12 to 15, ≥16 years old, the season of flushing (‘Season’) with 36 classes representing different months and the treatment of the donor mare. Seven different treatment classes (‘TC’) are defined based on the last and penultimate treatment prior to flushing. Mares having received no treatment at all belong to class ‘0’. The remaining mares are grouped into animals whose last treatment was a heat inducing agent (classes ‘1’ to ‘3’) or an ovulation inducing agent (classes ‘4’ to ‘6’). The further classification is done based on the penultimate treatment within 28 days before flushing, which can be none (classes ‘1’ and ‘4’), a heat inducing agent (classes ‘2’ and ‘5’) or an ovulation inducing agent (classes ‘3’ and ‘6’). TC 3 (last treatment = induction of heat, penultimate treatment = induction of ovulation) was discarded due to a small number of observations (N = 30). The consecutive number of flushing within years (‘NOF’) was divided into 9 classes (1, 2, 35 3, 4, 5, 6, 7, 8, ≥9). The impact of these effects on OR and RE has been analyzed previously [18]; a more detailed description can be found there. A threshold mixed model with a probit link function as implemented in the GLIMMIX procedure of the SAS package [19] was applied to data analysis. For OR, the following model was used: Φ ##& , where is the expected probability to observe at least two ovulations and Φ is the cumulative probability function of the standard normal distribution. , , and represent the fixed effects of i-th age class (i = 1,...,7), the j-th month of flushing (j = 1,...,28), the k-th preparation of the donor (k = 1,...,4) and the lth flushing number within season (l = 1,...,9), respectively. ##& is the m-th reproductive status of the donor mare (m = 1,…,3). The random effect of the n-th donor mare is represented by (n = 1,...,506). Due to the reduced number of mares with complete information on reproductive history, the number of factor levels is reduced for some effects. The embryo recovery rate was analysed using the following model: Φ ' #$$% , where is the expected probability to obtain at least one embryo and Φ is the cumulative probability function of the standard normal distribution. ' stands for 36 the m-th investigated effect (m = 1,…,3), which can be the reproductive status of the donor mare (##& ), the sort of used semen (( ), the number of spermatozoa ()%$ ) or the motility ()# ). , , and represent the fixed effects of i-th age class (i = 1,...,7 for ‘Status’ and 1,…,8 for the other investigated effects), the j-th month of flushing (j = 1,...,28 for ‘Status’, 1,…,19 for ‘Semen’ and 1,…,15 for ‘Mil’ and ‘Mot’), the k-th preparation of the donor (k = 1,...4), the l-th flushing number within season (l = 1,...,9), respectively. The random effects of the n-th donor mare and the o-th service stallion are represented by (n = 1,...,506 for ‘Status’, 1,…,535 for ‘Semen’ and 1,…,409 for ‘Mil’ and ‘Mot’) and #$$% (o = 11,...,142 for ‘Status’, 1,…,93 for ‘Semen’ and 1,…,50 for ‘Mil’ and ‘Mot’), respectively. Due to a reduced number of complete records, the number of factor levels is reduced for some effects. The random effects of the donor mare and the stallion were analyzed previously [18] and only included here to correctly set up the model. The results were not further regarded in the current study. Results Different impacts of the donor mare’s reproductive status on the two traits OR and RE were observed. No effect was found for RE, while OR is significantly influenced (Figure 1). The probability for obtaining at least two ovulations is significantly lower in mares that have been pregnant in the previous season (0.17) as compared to mares that have been barren or donors (0.27). There are no significant differences between the groups regarding the probability of obtaining an embryo. However, there is a slight tendency that mares previously used as donors or pregnant in the last season have a higher probability to deliver an 37 embryo of 0.48 and 0.47, respectively, as compared to barren mares with a probability of 0.44. 0.35 0.6 b b Probability of recovering at least one embryo Probability of >1 ovulation 0.30 0.25 0.20 a 0.15 0.10 0.05 0.00 0.5 0.4 0.3 0.2 0.1 0.0 Pregnant Barren Donor Pregnant Reproductive status in last season Barren Donor Reproductive status in last season Figure 1 Probability of more than one ovulation and recovering at least one embryo, respectively, conditional on mare’s reproductive status in the previous breeding season (F-test: 0.0001; LS-Means). The type of semen has a strong impact on RE (Figure 2). The probability for recovering at least one embryo when using fresh semen (0.52) differs significantly from the probability when inseminating with frozen semen from one (0.43) or different ejaculations (0.36). No significant difference can be found between the two types of frozen semen. 38 0.7 Probability of recovering at least 1 embryo 0.6 a 0.5 b b 0.4 0.3 0.2 0.1 0.0 Fresh Frozen from single ejaculation Frozen from different ejaculations Type of semen Figure 2 Probability of recovering at least one embryo conditional on the type of semen (F-test: 0.0001; LS-Means) The number of spermatozoa per insemination showed no significant effect on RE. The use of 120 to 200 million spermatozoa per insemination resulted in a probability of 0.57 to recover an embryo, while the other classes (≤100 mio and ≥250 mio) had an equal probability of 0.51. The motility of the spermatozoa on the other hand exhibited a significance influence on the recovery rate (Figure 3). Semen with a motility greater than 65% resulted in a significantly higher probability to obtain an embryo (0.59) than semen with a motility of 50 to 60 % (0.52) and semen with a motility less than 45% (0.47). 39 0.7 b a Probability of recovering at least 1 embryo 0.6 a 0.5 0.4 0.3 0.2 0.1 0.0 ≤ 45% 50−60% ≥ 65% Proportion of motile spermatozoa Figure 3 Probability of recovering at least 1 embryo depending on the proportion of motile spermatozoa in fresh semen (F-test: 0.0001; LS-Means) The fixed effects of the donor mare’s age, the season, the treatment of the donor and the consecutive number of the flushing have previously been analyzed [18]. It was shown that all effects significantly influence OR. All effects except for the number of flushings exhibited a significant influence on RE. These results were confirmed within the current study with two exceptions, which are probably due to the reduced sample size: the effect of flushing number was not significant for OR and the treatment was not significant for RE. Discussion A successful flushing requires a successful insemination. Vidament et al. [2] reported that the breeding status and the management of the mare during the estrus period as 40 well as the quality of the semen are the most important factors for achieving a pregnancy, especially when using frozen semen. Furthermore, numerous other studies have shown that the reproductive history is one of the main factors that have to be considered when using a mare as a donor for embryo transfer [3-7]. The number of embryos decreased with a poor reproductive history and higher ages of the donor mare. Within the current study, a significant influence of the mare’s breeding status on OR was found, while RE did not significantly differ between groups. The OR was significantly lower if the donor mare was pregnant in the previous breeding season. Stress resulting from the foal at foot and lactation possibly reduces OR, which could be expected to reduce RE in an equivalent way. This was, however, not observed. A reduced OR combined with an unaltered RE means that the fertilization rate and/or the survival rate of the early embryo is higher in those mares. A possible explanation might be a randomly increased use of fresh semen in the mares that were pregnant in the previous season. This would lead to an increased embryo recovery as discussed below. In fact, ~65% of those mares were inseminated with fresh semen compared to ~58% in the other groups, which might partly explain this observation. The type and the quality of semen also affected embryo recovery. The use of fresh semen resulted in a probability to recover an embryo of 0.52 was thus clearly superior to the use of frozen semen, which resulted in probabilities of 0.43 (1 ejaculation) and 0.36 (>1 ejaculation). One explanation for the reduced pregnancy rates after insemination with frozen semen was suggested by Dobrinski et al. [20] and Ellington et al. [21]. They demonstrated an impaired adhesion of previously frozen spermatozoa to the oviductal epithelium. This reduces the ability of the mare to create reserves of spermatozoa within the reproductive tract. Since insemination usually takes place prior to ovulation, the adhesion ensures the presence of viable 41 spermatozoa at the time of ovulation. As a consequence, the predictability of ovulation is crucial when using frozen semen requiring dense ultrasonographic monitoring and/or the use of an ovulation inducing agent. Alternatively, several consecutive inseminations can be performed, which increases the costs and reduces the number of potential offspring from a given stallion. It is known that there is a variation in semen freezability between different stallions as well as between different ejaculations from the same stallion. Thus, pregnancy rates obtained with frozen semen in commercial programs were reported to vary in wide range from 32 to 73% per cycle [12]. When there are large differences between different ejaculations from the same stallion, the probability of failure for a given dose of frozen semen can be reduced by pooling semen from different ejaculations. Within this study, no significant differences were observed between single ejaculation and pooled semen. There is, however, a tendency that doses from a single ejaculation resulted in a higher embryo recovery rate. This might be due to the different handling of pooled semen or the fact that pooling is preferably done in stallions with poor or rather varying freezability. For those inseminations performed with fresh semen, data on the number and motility of spermatozoa were available. Motility showed a significant influence on the embryo recovery rate, while the number of spermatozoa did not exhibit a significant effect. Even for doses with less than 100 mio spermatozoa the probability to recover an embryo was 0.51. The usage of a much smaller dose might result in inferior recovery rates, but this was naturally not attempted. For doses with a satisfying quality there is no improvement above a certain number of spermatozoa. For frozen semen, the impact of dose size might be more pronounced. The use of highly motile spermatozoa (≥65%) resulted in at least one embryo in 59% of cases, whereas the use of spermatozoa with a lower motility (≤45%) resulted in a success rate of 47%. Hence, the important fact for recovering at least one embryo is 42 the use of high motile spermatozoa (≥65 %) for insemination. This is in agreement with the results of Varner and Blanchard (unpublished) reported by Brinsko [22]. They found a pregnancy rate per cycle of only 48% for fresh semen, when the progressive sperm motility was <60%. In the case of using >60 % motile sperm, the pregnancy rate per cycle was 67%. Notably, we did not find the previously reported influence of the donor mare’s treatment when analyzing the fresh semen characteristics. This is probably due to the fact that the fresh semen can survive for a longer time in the female oviduct making the time span between insemination and ovulation less important. Conclusion Although the breeding history of the donor mare influences the ovulation rate, no differences in embryo recovery were found between groups with different histories within the current study. The use of fresh semen leads to higher embryo recovery rates than the insemination with frozen semen. Moreover, the motility of spermatozoa significantly influenced the number of recovered embryos. When using frozen semen, the treatment of the donor mare is of great importance to optimally determine the time of ovulation. Acknowledgments The authors thank the H. Wilhelm Schaumann Stiftung, Hamburg, for the financial support of H. von der Ahe. 43 References 1. Von der Ahe H, Stamer E, Kalm E, Thaller G, Tetens J. Analysis of genetic and management factors affecting ovulation and embryo recovery rate in German Warmblood donor mares. Theriogenology 2013. 2. Vidament M, Dupere AM, Julienne P, Evain A, Noue P, Palmer E. Equine frozen semen: Freezability and fertility field results. Theriogenology 1997;48: 907-917. 3. Squires EL, Imel KJ, Iuliano MF, Shideler RK. Factors affecting reproductive efficiency in an equine embryo transfer programme. J Reprod Fertil Suppl 1982;32: 409-414. 4. McKinnon AO, Squires EL, Voss JL, Cook VM. Equine embryo transfer. Compendium on Continuing Education for the Practicing Veterinarian 1988;10: 343-355. 5. Vogelsang SG, Vogelsang MM. Influence of donor parity and age on the success of commercial equine embryo transfer. Equine Veterinary Journal 1989;21: 71-72. 6. Squires E, Seidel G. Collection and transfer of equine embryos. Animal Reproduction and Biotechnology Bulletin No 11 Fort Collins CO: Colorado State University 1995: 7-9; 11-15; 27-32. 7. Carnevale E, Ginther O. Defective oocytes as a cause of subfertility in old mares. Biol Reprod Monogr 1 (Equine Reproduction VI) 1995: 209-214. 8. Ball BA, Little TV, Weber JA, Woods GL. Survival of day-4 embryos from young, normal mares and aged, subfertile mares after transfer to normal recipient mares. Journal of Reproduction and Fertility 1989;85: 187-194. 9. Samper JC. Management and fertility of mares bred with frozen semen. Animal Reproduction Science 2001;68: 219-228. 44 10. Samper JC, Hearn PH, Ganheim A. Pregnancy rates and effect of extender and motility and acrosome status of frozen-thawed stallion spermatozoa. Proceedings of the 40th Ann. Conv. Amer. Assoc. Equine Pract., 1994;41-43. 11. Sanchez R, Gomez I, Samper JC. Artificial insemination with frozen semen. In: Samper JC (ed), Equine Breeding Management and Artificial Insemination 2nd Revision edition edition: Saunders, 2008;175-185. 12. Loomis PR. The equine frozen semen industry. Animal Reproduction Science 2001;68: 191-200. 13. Klug E, Treu H, Hillmann H, Heinze H. Results Of Insemination Of Mares With Fresh And Frozen Stallion Semen. Journal of Reproduction and Fertility 1975: 107-110. 14. Tischner M. Results Of Artificial-Insemination Of Horses In Poland In Post-War Period. Journal of Reproduction and Fertility 1975: 111-114. 15. Salazar-Valencia F. Embryo recovery rates in mares of the pasofino Colombiano breed and deep freezing stallion semen in the tropics. Theriogenology 1983;19: 146-146. 16. Palmer E, Magistrini M. Automated analysis of stallion semen post-thaw motility. Acta veterinaria Scandinavica Supplementum 1992;88: 137-152. 17. Samper JC, Hellander JC, Crabo BG. Relationship between the fertility of fresh and frozen stallion semen and semen quality. Journal of reproduction and fertility Supplement 1991;44: 107-114. 18. Von der Ahe H, Tetens J, Stamer E, Kalm E, Thaller G. Analysis of genetic and management factors affecting ovulation and embryo recovery rate in German Warmblood Donor Mares. Theriogenology 2013. 19. Inc. SI. SAS, SAS 9.2 TS Level 1 M0 edition. Cary, NC, USA, 2008. 45 20. Dobrinski I, Thomas PGA, Ball BA. Cryopreservation reduces the ability of equine spermatozoa to attach to oviductal epithelial cells and zonae pellucidae in vitro. Journal of Andrology 1995;16: 536-542. 21. Ellington JE, Samper JC, Wright WR. Stallion sperm interactions with oviduct cells in vitro as an indicator of field fertility. Proceedings the 14th International Congress Animal Reproduction, Stockholm, Sweden, 2000;276. 22. Brinsko SP. Insemination doses: How low can we go? Theriogenology 2006;66: 543-550. 46 Chapter Three: Effects on success of pregnancy rate after embryo transfer in warmblood recipient mares and comparison of embryo transfer foals to non – embryo transfer foals H. von der Ahe*, E. Stamer†, J. Tetens*, E. Kalm* and G. Thaller* * † CAU, Institute of Animal Breeding and Husbandry, Hermann-Rodewald-Straße 6, 24098 Kiel, Germany TiDa GmbH, Bosseer Str. 4c, 24259 Westensee/Brux, Germany 47 ABSTRACT Considering the procedure “equine embryo transfer” there are several points where a decrease of the success rate, due to different influencing factors, is possible. The result of the first pregnancy determination after embryo transfer in the recipient mare is influenced by the season, the preparation of the recipient mare and the age of the embryo at time of transfer. Comparing embryo transfer foals to non – embryo transfer foals, there are only differences in the length of gestation. However, the gender of the foal, the age of the mare at time of birth and the season influences analyzed traits, like length of gestation, withers height at birth and weight at birth. Keywords: horse, ET, German Warmblood, foal, recipient INTRODUCTION The management of recipients is the main factor affecting the success of equine as well as bovine embryo transfer for example. Generally, recipients should show normal cyclic activity and abnormalities or pathological findings of the genital tract should be absent (Squires et al., 1999). Furthermore, the ultrasonographic monitoring of ovulation and uterine changes is of great importance. Estrus synchrony of donor and recipient mares substantially affects the success of embryo transfer, i.e. the pregnancy rate (Squires et al., 1982; Squires et al., 1985; Squires et al., 1985; McKinnon et al., 1988; Squires et al., 1995). Due to individual variation of the heat length, the exact timing of ovulation in donor and recipient is, however, difficult. An approach to handle this is the employment of induced ovulation in the recipient once an ovulation has been detected in the donor mare. The highest initial pregnancy rates of >75% at day 12 and around 65% at day 50 will be achieved when the recipient mare’s ovulation occurs within a time range of two days before to 48 three days after the donor mare’s ovulation (McKinnon et al., 1988; Carney et al., 1991). The decrease in pregnancy rates of more than ten percent between day 12 and 50 is similar to the amount of early embryonic loss of 13.4% found in mares inseminated with fresh semen (Carney, et al., 1991) and also, comparable results have been reported by Villahoz et al., (1985). Considering the method of transfer, there is a difference between surgical and nonsurgical transfer. Some investigators found lower pregnancy rates after performing nonsurgical transfer (27% vs. 53%, Imel et al., 1981; 45% vs. 72%, Iuliano et al., 1985). Other authors reported acceptable pregnancy rates after nonsurgical transfer like 75 to 77% (Hinrichs and Kenney, 1987; Wilson et al., 1987). As a result of its simplicity and in terms of animal welfare, the nonsurgical transfer is actually the most common technique. Additionally, the quality of the transferred embryo will affect the pregnancy rate. Equine embryos can be graded applying an approach similar to the procedure used in bovine embryos. Using fresh high quality embryos will then result in significantly higher pregnancy rates. A day 12 pregnancy rate of 70 to 75% can be achieved by using high quality embryos, followed by a day 50 pregnancy rate of 65% (Squires et al., 2002). The transfer of large embryos will on the other hand reduce the pregnancy rate (Squires et al., 1985 [No. 11]; Iuliano et al., 1985). Size of the recipient mare in comparison to donor mare and service stallion is an aspect which is often discussed. Studies in Newmarket (Great Britain) where Thoroughbred embryos were transferred to pony recipient mares and vice versa showed an imbalance between the genetically determined size of the embryo and recipient mare’s influence on intrauterine and postnatal development. Transfer into smaller recipients resulted in a retardation of fetus growth, which could not totally compensated (Allen et al., 2004). Thoroughbred foals that were transferred to a pony 49 recipient mare, showed signs of immaturity after birth (Ousey et al., 2004). In general, the transfer of warmblood embryos into trotter or thoroughbred mares, which had commonly a similar size can sometimes lead to a slightly reduced size at birth, but this is fully compensated during postnatal development. Satuè et al., (2011) reported a mean gestational length of 332.4 ± 12.1 days in Carthusian broodmares, which was significantly prolonged (5.7 days) when mares give birth to colts (336.8 ± 11.0 days) rather than fillies (331.1 ± 12.8 days). They also found a 5.3 days shorter pregnancy in mares 8 to 12 years (330.8 ± 11.0 days) old in contrast to those of 13 to 17 years (336.1 ± 8.9 days). Valera et al., (2006) found mean length of gestation of 336.8 ± 0.48 days in Andalusians or Spanish Purebreds and 340.3 ± 0.63 days in Arabian mares. They found that two of the main factors significantly affecting gestational length are the age of the mother and the sex of the foal. Cilek (2009) investigated Purebred Arabian foals and found a significant influence of sex of the foal on the birth weight. The weight at birth was 46.27 ± 0.40 kg for colts, whereas fillies weighted 45.22 ± 0.40 kg at birth. Allen et al., (2002) found a mean duration of gestation of 339.0 ± 3.0 days in Thoroughbred mares. The mean foal birth weight was 53.1 ± 2.6 kg. MATERIAL AND METHODS The data set comprised 3 130 embryo transfers performed between 2004 and 2008 on a large stud farm in Northern Germany. 910 recipient mares were used for embryo transfer in this period. The data set was reduced due to missing information (e.g. birth date of recipient mares), resulting in 2 927 evaluable transfers. Frozen embryos were also excluded, because only 59 of these embryos were transferred in the period 50 from 2004 to 2008. Table 1 gives an overview of the number of embryo transfers per year. Table 1 Number of embryo transfers per year (2004 to 2008) year performed embryo transfers [n] 2004 552 2005 549 2006 536 2007 605 2008 685 total 2 927 The number of transfers per recipient lay in a range of 1 to 11 with an average of 3.4 transfers per recipient mare over the five year period. The number of transfers within years and recipient mares was between one and four, with an average of 1.4 transfers per recipient and year. Reproduction data of recipient mares were registered daily by the veterinarians and promptly stored on record sheets. The description of the results of pregnancy determination (PD) is shown in Table 2. 51 Table 2 Description of pregnancy determination (PD) at different times after ovulation (2 927 transfers) positive (n) positive (%) PD I (16 d after ovulation) 1955 66.8 PD II (45 d after ovulation) 1706 58.3 PD III (150 d after ovulation) 1543 52.7 d = days The analyzed reproduction trait is PD I of the recipient mare around 9 days after transfer (Table 3). PD I was defined as a binary trait: in the case of a negative PD I result the binary code is 0, and in the case of a positive PD I result, the code is 1. Table 3 Description of analyzed trait first pregnancy determination (PD I), 16 d after ovulation binary code 0 trait pregnancy determination (PD I) 1 n transfers n % 2 927 972 33.2 n % 1955 66.8 Influencing factors: Age classes of recipient mare’s age at time of transfer were defined as 2, 3, 4, 5, 6, 7, 8, 9, 10+11, 12+13, ≥ 14 years old. The average age of recipient mares at time of transfer was 7.5 years within the five year period. Generally, the transfer season ranged from March to September. Number of average transfers per month was 88.8 over the five year period. 52 The treatment code (TC) represented six different combinations between point of treatment before ovulation (last and second last treatment) and type of heat and ovulation inducing agents (Table 4). TC 3 was discarded due to small number of observations (n = 50). Table 4 Definition of treatment code (TC) last treatment second last treatment TC none none 0 heat inducing agent none 1 heat inducing agent heat inducing agent < 28 d 2 heat inducing agent heat inducing agent > 28 d 1 heat inducing agent ovulation inducing agent < 28 d 3 heat inducing agent ovulation inducing agent > 28 d 1 ovulation inducing agent none 4 ovulation inducing agent heat inducing agent < 28 d 5 ovulation inducing agent heat inducing agent > 28 d 4 ovulation inducing agent ovulation inducing agent < 28 d 6 ovulation inducing agent ovulation inducing agent > 28 d 4 *d=day Embryos were flushed and transferred at an age lying in the range of 6 to 9 days. The synchrony between the donor and recipient mare regarding the day of ovulation was divided into six classes. The difference of time of ovulation ranged from 0 days up to 5 days. Significance of fixed effects was analyzed using the GLIMMIX procedure of the SAS package (SAS 2008) with a threshold mixed model and a probit link function: Pregnancy determination [model]: E [πijklmnop] = Φ (Agei + Seasonj + TCk + AgeEmbl + Synm + recipn + donoro + stallionp) 53 where E [πijklmnop] = Expected probability to obtain a positive PD Φ = Cumulative probability function of standard normal distribution Agei = Fixed effect of i-th age (i = 1,...,11) Seasonj = Fixed effect of the j-th month of transfer within year (j = 1,..., 33) TCk = Fixed effect of the k-th treatment of the donor (k = 1,..., 6) AgeEmbl = Fixed effect of the l-th age of the embryo (l = 1,..., 4) Synm = Fixed effect of the m-th synchrony of day of ovulation (m = 1,..., 6) recipn = Random effect of the n-th recipient mare (n = 1,..., 861) donoro = Random effect of the o-th donor mare (o = 1,..., 586) stallionp = Random effect of the p-th service stallion (p = 1,...,152) The data set of the ET and Non-ET foals contained 4 288 records. The foals descended from 192 sires. Table 5 gives an overview of foals born from 2005 to 2009. Table 5 Distribution of number of foals born between 2005 and 2009 year born foals [n] 2005 735 2006 916 2007 886 2008 922 2009 829 total 4 288 54 Due to missing data of the pedigree 4 268 foals were analyzable. The number of analyzable ET foals was 1 611 (37.8 %), whereas the number of Non-ET foals were 2 657 (62.2 %). Analyzed traits are the gestational length (GL), withers height at birth (HB) and the weight at birth (WB). The data sets contained 3 436 records, 2 283 records and 1 322 records for gestational length, withers height at birth and weight at birth, respectively. The fixed effect ‘ET’ was divided into two classes, embryo transfer foals and non embryo transfer foals. The fixed effect ‘Sex’ represented two classes, colts and fillies. The fixed effect ‘Season’ represented the month of birth of the foal and was only used for analyzing gestational length. The fixed effect ‘Age_moth’ was the age of the recipient mares as well as of the mares, who were pregnant with their own foals. The models were analyzed using the SAS procedure MIXED and the REML method (SAS Institute, 2008). The resulting model for the analysis of the data sets was yijklmn = µ + ETi + Sexj + Seasonk +Age_mothl + stallionm + eijklmn where yijklmn = n-th observation of the analyzed trait (GL/HB/WB) µ = overall mean ETi = Fixed effect of the i-th type of foal (ET/Non-ET) (i = 1, 2) Sexj = Fixed effect of the j-th gender of the foal (j = 1, 2), Seasonk = Fixed effect of the k-th month of birth of the foal (k = 1,…, 27 for GL), Age_mothl = Fixed effect of the l-th Age of the mother at time of birth (l = 1,…,12 for GL, l = 1,…, 11 for HB, l = 1,…, 6 for WB) 55 stallionm = Random effect of the m-th service stallion (m = 1,…, 159 for GL, m = 1,…, 146 for HB, m = 1,…, 118 for WB) eijklmn = Random residual effect. RESULTS The 3 130 performed transfers resulted in 1 611 living born foals, which is a success rate of 51.5 %. After PD I, 66.7 % of the pregnancy determinations were positive. The failure of pregnancy after PD I was quite low, with 94.5 % and 97.7 % positive results at PD II and PD III, respectively. The following fixed effects were investigated in terms of affecting PD I around nine days after transfer to the recipient mare. Month of transfer (Season), preparation of the recipient mare (TC) and the age of embryo at time of transfer (AgeEmb) showed a significant influence on PD I. The age of the recipient mare at time of birth (Age_moth) and the synchrony of donor and recipient mare regarding the day of ovulation (Syn) showed no significant influence on the result of PD I. Regarding the season, number of positive pregnancy results (PD I) were high in spring and early summer, but generally decreased in midsummer. The probability (and standard error) to obtain a positive PD I ranged from 0.46 ± 0.05 (June 2008) to 0.86 ± 0.03 (May 2004). Pregnancy rates were generally lower in 2008 than in other years. The years 2005, 2007 and 2008 showed also higher pregnancy rates in late summer and autumn. The probability (and standard error) of obtaining a positive PD I with regard to the preparation of the recipient mare (Figure 1) was between 0.61 ± 0.02 (for TC = 0) and 0.75 ± 0.04 for (TC = 6). Only TC = 0, representing recipient mares, that got no treatment at all, showed a significant lower probability for a successful PD I. Between 56 classes 1 and 2 (last application was a heat inducing agent) and classes 4, 5 and 6 (last application was an ovulation inducing agent) no significant differences were detected. b b b b b a a+b different superscripts indicate significant differences in least square means (P<0.05), according to Bonferroni correction Figure 1 Probability of obtaining a positive pregnancy determination (PD I) depending on the treatment of the recipient mare (TC) before transfer (F-test: 0.0001; LS-Means) The age of the embryo at time of transfer played an important role. The probabilities (and standard error) of obtaining a positive PD I were between 0.62 ± 0.03 (Day 9 old embryos) and 0.74 ± 0.02 (Day 7 old embryos). Whereas there is no significant difference regarding PD I between Day 7 and Day 8 embryos, 9 day old embryos showed a significant lower probability for a positive PD I (Figure 2). Day 6 embryos did not differ significantly from seven, eight or nine day old embryos with respect to the PD I. 57 a,b a+b a a b different superscripts indicate significant differences in least square means (P<0.05), according to Bonferroni correction Figure 2 Probability of obtaining a positive pregnancy determination (PD I) depending on the age of embryo (d=days) at time of transfer (F-test: 0.0001; LS-Means) The age of the recipient mare at time of transfer showed no significant influence on PD I, although there was a slight tendency for better pregnancy results in younger mares (two to seven years old). The probability (and standard error) of obtaining a positive PD I ranged from 0.63 ± 0.03 (recipient mares ≥ 14 years old) to 0.72 ± 0.03 (recipient mares that were 2 and 6 years old), respectively. The synchrony, defined as the difference of days of ovulation of donor mare and recipient mare, had no significant influence on PD I. The probability (and standard error) for a positive PD I were in a range of 0.67 ± 0.03 (one day difference) to 0.72 ± 0.04 (five days difference). Within the years 2005 to 2009 the number of living born foals was 4 288, divided into 1 611 ET foals and 2 657 Non-ET foals. Table 6 show the results of gestation length 58 (GL), height at birth (HB) and weight at birth (WB) compared as ET and Non-ET foals (= type of foals) as well as male and female foals. Table 6 Values and standard errors [se] of the traits gestation length, height at birth and weight at birth regarding to ET and Non-ET foals as well as to male and female foals Gestation length Height at birth Weight at birth (days) [se] (centimeter) [se] (kilogram) [se] ET 333,1a [0.35] 104,2 a [0.15] 57,3 a [0.31] Non-ET 333,8b [0.32] 104,4 a [0.14] 57,7 a [0.26] male 334,5a [0.33] 104,6a [0.15] 58,2a [0.28] female 332,5b [0.33] 103,9b [0.15] 56,8b [0.28] a+b different superscripts indicate significant differences in least square means (P<0.05), according to Bonferroni correction The length of gestation after ET was significantly shorter than those in mares who were pregnant with their own foals. Withers height at birth and weight at birth showed no significant differences between ET and Non – ET foals. All three traits were significant influenced by the gender of the foal. Length of gestation in colts was two days longer than fillies. Colts were 0.7 centimeters taller than fillies. Weight at birth in colts was 1.4 kilogram higher than in fillies. There was no interaction between the type of the foal (ET/Non-ET) and the gender of the foal. Regarding the age of the mare at time of birth, there was a significant influence on gestational length (Figure 3), on withers height at birth (Figure 4) and weight at birth (Figure 5). Values for gestational length were between 331.9 d (age class = 5) and 336.5 d (age class = 17). Standard errors lay in a range of 0.41 (age class = 3) and 59 0.70 (age class = 11). Mares at an age of ≥17 years showed a higher length of gestation than younger mares. Furthermore, mares at an age of three to seven years old were significantly shorter in foal than mares at an age of 11 to 16 years old. 338 c Length of gestation (d) 337 336 b 335 334 333 a a a 3 4 a a,b a,b b b a,b a 332 331 330 a-c 5 6 7 8 9 10 Age of (recipient) mare 11 12 14 17 different superscripts indicate significant differences in least square means (P<0.05), according to Bonferroni correction Figure 3 Influence of the age of the mare on the trait gestational length (F-test: 0.0001; LS-Means) Withers height at birth was also significantly influenced by the age of the mare at time of birth. Values for withers height at birth were between 101.2 cm (age class = 3) and 105.6 cm (age classes = 9 and 10). Standard errors lay in a range of 0.20 (age classes = 3 and 4) and 0.34 (age class = 10). Younger mares (≤7 years old) and mares older than 13 years gave birth to smaller foals. 60 Withers height at birth (cm) e 106 105 c,g 104 c,f g a 101 100 3 a-g d,f e e,f b 103 102 f,g e 4 5 6 7 8 9 10 Age of (recipient) mare 11 13 16 different superscripts indicate significant differences in least square means (P<0.05), according to Bonferroni correction Figure 4 Influence of the age of the mare on the trait height at birth (F-test: 0.0001; LS-Means) The age of the mare at time of birth showed also a significant influence on weight at birth. Values for weight at birth were between 53.0 kg (age class = 3) and 60.2 kg (age class = 9). Standard errors lay in a range of 0.40 (age class = 3) and 0.46 (age class = 6). Mares, younger than six years old, gave birth to foals with lighter weights. Although there was no significant difference between the age classes 9 and 12; mares, older than eleven years showed a tendency to bear foals with slighter weights at birth. 61 62 d d c,d 60 Weight at birth (kg) c 58 b 56 54 a 52 50 3 a-d 4 6 8 Age of (recipient) mare 9 12 different superscripts indicate significant differences in least square means (P<0.05), according to Bonferroni correction Figure 5 Influence of the age of the mare on the trait weight at birth (F-test: 0.0001; LS-Means) For analyzing gestational length the model contained additionally the fixed effect season, which was the month of birth of the foal (Figure 6). The season showed a significant effect on gestational length. Values for gestational length regarding the season were between 326.3 d (May 2007) and 340.1 d (February 2009). Standard errors lay in a range of 0.69 (May 2009) and 1.2 (February 2006 and January 2008). 62 342 340 Length of gestation (d) 338 336 334 332 330 328 326 324 322 320 2005 2006 2007 2008 2009 Month of birth Figure 6 Influence of the season on the trait length of gestation (F-test: 0.0001; LSMeans) DISCUSSION There is a large variation in pregnancy rates after nonsurgical transfer, ranging from 50 to 75 % (Squires et al., 1995; Mc Kinnon et al., 1988; Riera et al., 1993; Vogelsang et al., 1985). This is in agreement with our results showing a positive result of PD I of 66.7 % and a success rate of 51.5% regarding the living born foals. Considering the method of transfer, there is a difference between surgical and nonsurgical transfer. But there were also lower pregnancy rates found after performing nonsurgical transfer (27% vs. 53%, Imel et al., 1981; 45% vs. 72%, Iuliano et al., 1985) as well as other authors reported acceptable pregnancy rates after nonsurgical transfer like 75 to 77% (Hinrichs and Kenney, 1987; Wilson et al., 1987). 63 No significant differences in PD I were detected depending on the age of the recipient mare at time of transfer. This is in contrast to the influence of the age on embryo recovery rate in donor mares founded in previous studies, where mares older than 16 years donate fewer numbers of embryos. Hence, the ability to become pregnant is independent of the age of the recipient mare in contrast to the production of an embryo, which depends on the age of the donor mare. With respect to the season, there is a significant influence on PD I. Higher numbers of positive PD I were achieved in spring and early summer, but generally decreased in midsummer. In spite of the fact, there is no clear trend between the years. It is generally known that the reproduction cycle of horses starts naturally when daylight is extending. Hence, spring and early summer are the best time to achieve acceptable pregnancy rates after embryo transfer. For the years 2005, 2006 and 2008 we found a rise of positive pregnancy determinations in late summer and autumn. This increase may be depending on the weaning of the foals, because mares with no foal at foot are often more likely to become pregnant. The treatment of the recipient mare differs only between recipient mares that got no treatment at all and processed mares independent of the treatment were heat inducing agents or ovulation inducing agents. Treated mares showed higher numbers of positive PD I than untreated mares. The management of the recipient mare plays an important role and therefore the timing and detection of the ovulation is of great importance for the success of embryo transfer. In treated mares this is more predictable than in untreated mares. With a view to the age of the embryo at time of transfer, best pregnancy rates were obtained when transferring Day 7 embryos or Day 8 embryos. Day 9 embryos resulted in significant lower pregnancy rates due to potential damage during the flushing and handling before transfer. This result is in agreement with Squires et al. 64 [11], (1985) and Iuliano et al., (1985), who reported reduced pregnancy rates after the transfer of large embryos. Day 6 embryos showed no significant differences regarding the pregnancy rate compared to day 7, day 8 and day 9 embryos, which is an important result with view to frozen embryos (flushing at day 6 after ovulation). Especially for frozen embryos the viability and therefore acceptable pregnancy rates plays an important role, because of the forces of the freezing and thawing process. Estrus synchrony of donor and recipient mares substantially affects the success of embryo transfer, i.e. the pregnancy rate (Squires et al., 1982; Squires et al., 1985; Squires et al., 1985; McKinnon et al., 1988; Squires et al., 1995). Due to individual variation of the heat length, the exact timing of ovulation in donor and recipient is, however, difficult. An approach to handle this is the employment of induced ovulation in the recipient once an ovulation has been detected in the donor mare. The highest initial pregnancy rates of >75% at day 12 and around 65% at day 50 will be achieved when the recipient mare’s ovulation occurs within a time range of two days before to three days after the donor mare’s ovulation (McKinnon et al., 1988; Carney et al., 1991). Although this results have been found previously, in our study no significant influence of synchrony of donor and recipient could be detected. Recipient mares ovulated in a range of zero days up to five days after the donor mare, followed by comparable pregnancy rates after embryo transfer. The decrease in pregnancy rates of more than ten percent between day 12 and 50 is similar to the amount of early embryonic loss of 13.4% found in mares inseminated with fresh semen (Carney, et al., 1991) and also, comparable results have been reported by Villahoz et al., (1985). No value for the covariance parameter estimates could be found for the recipient mare in consideration of the first pregnancy determination. This indicates that there is no variation between different recipient mares. 65 Foals The comparison of embryo transfer and non embryo transfer foals showed only a significant influence with respect to the length of gestation. Comparing colts and fillies revealed in significant differences in all analyzed traits. No interaction was found between the type of the foal (ET foal/Non – ET foal) and the gender. Valera et al., (2006) found mean length of gestation of 336.8 ± 0.48 days in Andalusians or Spanish Purebreds and 340.3 ± 0.63 days in Arabian mares. They found that two of the main factors significantly affecting gestational length are the age of the mother and the sex of the foal. Allen et al., (2002) found a mean duration of gestation of 339.0 ± 3.0 days in Thoroughbred mares. Satuè et al., (2011) reported a mean gestational length of 332.4 ± 12.1 days in Carthusian broodmares, which was significantly prolonged (5.7 days) when mares give birth to colts (336.8 ± 11.0 days) rather than fillies (331.1 ± 12.8 days). In our study we found a longer length of gestation for colts of 2 days. The range is not that big as in the study above, but showed a significant longer length of gestation for colts rather than fillies as well. Satuè et al., (2011) also found a 5.3 days shorter pregnancy in mares 8 to 12 years (330.8 ± 11.0 days) old in contrast to those of 13 to 17 years (336.1 ± 8.9 days). This is also in agreement with our results, that mares (>10 years old) are significantly longer in foal than younger mares. Younger mares (≤7 years old) exhibit a significantly shorter gestational length (- 4 days) than mares older than 17 years. The comparison of ET and Non – ET foals resulted in a difference of gestational length. ET foals showed a shorter length of gestation than Non – ET foals. Although there are high standard errors in the present study, it is obvious, that the season (= month of birth) shows a significant influence on length of gestation, which decreases, when the foals are born in late summer or autumn. But there is no clear trend 66 between the years. Maybe there is a hormonal influence on the foal due to decreasing daylight, triggering the birth. Concerning withers height at birth no significant difference was detected between ET and Non – ET foals. However, there was a significant difference regarding the gender of the foal. Colts were 0.7 cm taller than fillies, maybe due to a significant longer length of gestation. Satuè et al., (2011) found also a significant influence of the mare’s age on withers height at birth, agreeing with our results, showing that younger mares (≤ 6 years old) and mares, older than 16 years, give birth to smaller foals. There is a significant influence of the mare’s age with an increasing height until the age of mare of 9, 10, 11, but decreasing in mares older than 11 years old. No significant influence could be detected between ET and Non-ET foals regarding withers height at birth as well as the weight at birth. With respect to the weight at birth Satuè et al., (2011) found younger mares (≤ 6 years) foaling offspring with little weights compared with mares older than eight years foaling heavier offspring. Cilek (2009) investigated Purebred Arabian foals and found a significant influence of gender of the foal on the birth weight as well. The weight at birth was 46.27 ± 0.40 kg for colts, whereas fillies weighted 45.22 ± 0.40 kg at birth. As reported by Elliot et al., (2009) who found a significant difference in birth weights of 1.1 kg between colts and fillies, we found results of higher birth weights in colts of 1.4 kg. Likewise, Elliot et al., (2009) found an increase of the weight at birth with an increase of the mare’s age up to 12 years. Compared to our results we found an increase of the weight at birth up to an age of the mare of 9 years, followed by a light decrease in weight at birth for older mares. Further research is requested to confirm these promising results. 67 CONCLUSION Considering the first pregnancy determination after embryo transfer in the recipient mare, significant effects of the season, the preparation of the recipient mare and the age of the embryo at time of transfer could be found. The comparison of embryo transfer foals and non – embryo transfer foals resulted in a significant difference concerning the length of gestation, but there was no significance detected for the traits withers height at birth and weight at birth. The age of the mare at time of birth played in important role, with regard to all three traits. Younger mares exhibited a shorter gestational length, a smaller withers height at birth and a lighter weight at birth of the foals. For the improvement of the procedure embryo transfer and the number of resulting foals, the management of the recipient is a useful approach in account with the season and the age of the embryo at time of transfer. No negative influences on vitality or other traits could be found in ET foals. A further step should be to monitor the development of the embryo transfer and non embryo transfer foals, regarding withers height, the weight and fertility, especially for embryo transfer foals. REFERENCES [1] Squires, E. L., McCue, P. M. and Vanderwall, D.; The current status of equine embryo transfer. Theriogenology 1999; 51: 91-104. [2] Squires, E. L. and Seidel Jr., G. E.; Collection and transfer of equine embryos. Animal Reproduction and Biotechnology Laboratory Bulletin No. 11. Fort Collins CO: Colorado State University, 1995; 7-9, 11-15, 27-32. [3] McKinnon, A. O., Squires, E. L., Voss, J. L. and Cook, V. M.; Equine embryo transfer: A review. Comp. Cont. Educ. Pract. Vet. 1988; 10: 343-355. 68 [4] Iuliano, M. F., Squires, E. L. and Cook, V. M.; Effect of age of equine embryos and method of transfer on pregnancy rate. Journal of Animal Science 1985; Vol. 60, No. 1: 258-263. [5] Squires, E. L., Carnevale, E. M., McCue, P. M. and Bruemmer, J. E.; Embryo technologies in the horse. Theriogenology 2002; 59: 151-170. [6] Satué, K., Felipe, M., Mota, J. and Muñoz, A.; Gestational length in Carthusian broodmares: effects of breeding season, foal gender, age of mare, year of parturition, parity and sire. Polish Journal of Veterinary Science 2011; Vol. 14, No. 2: 173-180. [7] Allen, W. R., Wilsher, S., Turnbull, C., Stewart, F., Ousey, J., Rossdale, P. D. and Fowden, A. L.; Influence of maternal size on placental, fetal and postnatal growth in the horse. I. Development in utero. Reproduction 2002; 123: 445453. [8] Valera, M., Blesa, F., Dos Santos, R. and Molina, A.; Genetic study of gestation length in andalusian and arabian mares. Animal Reproduction Science 2006; Vol. 95, Issue 1-2: 75-96. [9] Squires, E. L., Imel, K. J. and Iuliano, M.F.; Factors affecting reproductive efficiency in an equine embryo transfer program. J. Reprod. Fertil. 1982; 32(Suppl.): 409-414. [10] Squires, E. L., Garcia, R. H. and Ginther, O.J.; Factors affecting success of equine embryo transfer. Equine Vet. J. 1985; 3(Suppl.): 92-95. [11] Squires, E. L., Cook, V. M. and Voss, J. L.; Collection and transfer of equine embryos. Colorado State University Animal Reproduction and Biotechnology Lab. Bulletin 1985; 1: 16-25. [12] Carney, N. J., Squires, E. L., Cook, V. M., Seidel Jr., G. E. and Jasko, D. J.; Comparison of pregnancy rates from transfer of fresh versus cooled, transported equine embryos. Theriogenology 1991; Vol. 36, No. 1: 23-32. [13] Villahoz, M. D., Squires, E. L., Voss, J. L. and Shideler, R. K.; Some observations on early embryonic death in mares. Theriogenology 1985; 23: 915-924. [14] Allen, W. R., Wilsher, S., Tiplady, C. and Butterfield, R. M.; The influence of maternal size on pre- and postnatal growth in the horse: III Postnatal Growth. Reproduction 2004; 127: 67-77. [15] Ousey, J. C., Rossdale, P.D., Fowden, A. L., Palmer, L., Turnbull, C. and Allen, W. R.; Effects of manipulating intrauterine growth on post natal adrenocortical 69 development and other parameters of maturity in neonatal foals. Equine Vet. J. 2004; 36: 616-621. [16] Imel, K. J., Squires, E. L., Elsden, R. P., Shideler, R. K.; Collection and transfer of equine embryos. J. Amer. Vet. Med. Assoc. 1981; 179-987. [17] Hinrichs, K. and Kenney, R. M.; Effect of timing of progesterone administration on pregnancy rate after embryo transfer in ovariectomized mares. J. Reprod. Fertil. 1987; Suppl. 35: 439-443. [18] Wilson, J. M., Rowley, M. B., Rowley, W. K., Smith, H. A., Webb, R. L. and Tolleson, D. R.; Successful non-surgical transfer of equine embryos to postpartum mares. Theriogenology 1987; 27: 295. (Abstr.) [19] Cilek, S.; The survey of reproductive success in Arabian horse breeding from 1976-2007 at Anadolu State farm in Turkey. J. Anim. Vet. Adv. 2009; 8: 389396. [20] Riera, FL., and McDonough, J.; Commercial embryo transfer in polo ponies in Argentina. Equine Vet. J. 1993; 15(Suppl): 116-119. [21] Vogelsang SG, Bondioli, KR and Massey, JM.; Commercial application of equine embryo transfer. Equine Vet. J. 1985; (3): 89-91. [22] Elliot, C., Morten, J., Chopin, J.; Factors affecting foal birth weight in Thoroughbred horses. Theriogenology 2009; 71: 683-689. 70 General Discussion The aim of the present study was to determine which factors show a significant influence on the ovulation rate (OR), embryo recovery rate (RE) and the pregnancy rate in the recipient mare after embryo transfer. Furthermore the estimation of heritabilities for OR and RE should show whether the considering of fertility parameters in breeding programs makes sense. The comparison of embryo transfer foals and non embryo transfer foals should detect, whether embryo transfer foals have any disadvantages compared to non embryo transfer foals. In our study we achieved a success rate of 0.54 embryos per flush over all donor mares (712 mares). This value is in agreement with Douglas [2] (34% vs. 56%), Squires et al. [1] (28% vs. 80%) and Vogelsang and Vogelsang [24] (29% vs. 53 61%) who got probabilities to flush an embryo ranging from 0.28 to 0.34 in old or subfertile donor mares in contrast to a recovery rate of 0.53 to 0.80 in young fertile mares. Several summaries based on data from commercial operations have reported a 50 % embryo recovery rate from single – ovulating mares, some of which were subfertile mares [7,38]. There was a wide variety of donor mares at the stud, regarding the age and fertility. Hence, a success rate of 54% overall donor mares is a acceptable result. Despite horses belong to the monovular species, there are reports that multiple ovulations occur with frequencies ranging between 4 and 43 % [32,33]. In our study we found a probability to recover at least two ovulations of 1.22 ± 0.50, which was 22%. This is in agreement with the literature, where different percentage of multiple ovulations for different breeds are reported. The frequency of multiple ovulations in draft horses and Thoroughbreds ranges from 15 and 30 %, whereas that in light breeds and ponies showed a lower rate of around 2 to 10 % [32,33]. Another way to 71 increase the number of ovulation is the procedure of superovulation, but it is still being difficult and generally embryos result from only the normally developing one, or sometimes two, dominant follicles per cycle. With a view to cattle, successful superovulating in donor mares would highly increase the efficiency of equine embryo transfer. The main difficulty is that not every mare responds significantly to superovulation treatment such as immunization to inhibin, administration of pituitary extract or pulsatile gonadotropin releasing hormone. Today there is no product for superovulation that gains widespread acceptance for clinical use in equine embryo transfer. Thus, most of the mares in an embryo transfer program are currently not stimulated, and only single embryo collections are attempted. As shown above the probability for obtaining at least two ovulations were 22 % followed by a recovery rate of at least two embryos of 10%, so there is a loss of 12% between time of ovulation and flushing. Despite a higher ovulation rate in the donor mare, more than half of all ovulated ova do not result in an embryo after insemination. In most breeds, a low recovery rate due to the lack of multiple ovulation increases the time required to obtain a pregnancy in the recipient mare. This is associated with raising costs for the whole procedure of equine embryo transfer. Double or triple ovulations during a given cycle would result in higher embryo recovery rates than are currently possible from single – ovulating mares [21]. The age of the donor mare had a significant influence on the ovulation rate (OR). With an increasing age of the donor mare, the number of ovulations also increased. Donor mares >15 years showed a decreased probability to recover an embryo; those donor mares provide significantly smaller numbers of embryos. This is in agreement with Vogelsang and Vogelsang [24] who reported significant differences in embryo recovery rates in younger mares (2 to 8 and 9 to 17-year-old) compared with older mares (18 to 28-year-old), (80/132 [60.6%], 94/183 [51.4 %] and 93/309 [30.1%], 72 respectively). But there is an antagonism between OR and RE regarding the age of the donor mare: Although there is an increase of OR with an increased age of the donor mare, there is a lower RE at an increased age of the donor mare. Causes of reduced embryo recovery from these older mares included uterine and oviductal pathology, and increased early embryonic death [15]. Carnevale et al. [39] reported that defective oocytes from aged mares were a cause of reduced fertility. Vidament et al. [42] reported that the breeding status and the management of the mare during the estrus period as well as the quality of the semen are the most important factors for achieving a pregnancy, especially when using frozen semen. Furthermore many other studies found that the reproductive history is one of the main factors that have to be considered when using a mare as a donor mare for embryo transfer [1,19,24,39,38]. Number of embryos decreased with a poor reproductive history and higher ages of the donor mare. Samper et al., [44] reported that the number of older maiden mares (>8 years) with a prolonged sport or show career used for breeding is steadily increasing. Especially when bred with frozen semen, these mares had lower pregnancy rates due to histories of reproductive problems and were therefore potential candidates for failure. In our study, we generated three groups for the breeding status of the donor mare, showing a different influence on both traits (OR and RE). Whereas the ‘status’ was significant for the trait OR, there was no significant effect on RE. OR was significantly lower, when the donor mare was pregnant (‘status’ 1) before flushing. There was no significant effect on RE, but a slight positive tendency, that donor mares with ‘status’ 3 showed a higher probability to recover an embryo than the other ones. This suggests that a pregnancy before flushing influences a donor mare in a negative way regarding the number of ovulations. The use of donor mares two years in succession shows no negative effect concerning the reproductive performance of the mares. 73 Vanderwall [46] reported similar results with very high (>90%) fertilization rates in young mares under optimal conditions (i.e., fresh, fertile semen) versus older mares that showed a lower pregnancy rate of 85%. Although high fertilization rates were achieved, embryo recovery rates on days 6 to 9 after ovulation were distinctly lower for older mares [47,24], which indicates high embryonic losses during the first week of gestation. In other studies, the estimated embryonic loss rate between fertilization and day 14 was lower than 10% for young mares but up to 70% for older mares [48,15]. The type and the quality of semen also affect embryo recovery. The use of fresh semen resulted in a higher recovery rate compared to frozen semen. The fertility of (frozen) semen is influenced by several factors including semen quality, stallion, insemination dose, mare selection and mare management. It is well accepted that there is a variation in semen freezability among service stallions and further between ejaculates from the same stallion. Pregnancy rates obtained with frozen semen in commercial programs ranged from 32 to 73% per cycle [49] and from 26% [50,51] to 66% [52] per season with most values vary from 30 to 55% for the seasonal success rate [54,53,39]. The results of our study agreed with findings reported in the literature. For the use of fresh semen, we obtained a probability of 0.52 to recover at least one embryo, whereas probabilities for using frozen semen and frozen semen, which was gained on different dates were 0.43 and 0.36, respectively. This verifies a significant influence on the embryo recovery rate, regarding the type of semen used for insemination of the donor mare. For flushing donor mares, insemination with fresh semen is always preferable because of significant higher embryo recovery rates. But if fresh semen is not available it has to pay special attention to the management of the donor mare for the insemination with frozen semen to get acceptable results. 74 Considering the type of semen used for insemination, differences between fresh semen and frozen semen with respect to fertility could be found. A Disadvantage of frozen semen are lower fertility for many stallions [49]. The processing of the semen is often done by owners and not by professionals, which explained a wide variability in quality of the transported semen. In contrast to fresh semen, there is more technical expertise needed for processing frozen semen. Furthermore there are some stallions producing ejaculates, which are not suitable for cooling or storage. One explanation for the reduced pregnancy rate after insemination with frozen semen was reported by Dobrinski et al. [55] and Ellington et al. [56]. They demonstrated that frozen semen of some stallions does not bind to the oviductal epithelium of the mare in a normal way, reducing the ability of the mare to create reserves of spermatozoa within the reproductive tract. These reserves guarantee that survivable spermatozoa are available in the oviduct at the time of ovulation, when insemination happened before the mare ovulates. For investigating the number and motility of spermatozoa only fresh semen was considered, because there was no information about it for frozen semen. Motility showed a significant influence on the embryo recovery rate, whereas the number of spermatozoa did not show a significant influence. The use of high motile spermatozoa (≥65%) resulted in 59% in at least one embryo, whereas the use of spermatozoa with a lower motility (≤45%) resulted in a success rate of 47%. Hence, the important fact to recover at least one embryo is the use of high motile spermatozoa (≥65 %) for insemination. This is in agreement with the results of Varner and Blanchard (unpublished) reported by Brinsko [57]. They found a pregnancy rate per cycle of only 48% for fresh semen, when the progressive sperm motility was <60%. In the case of using >60 % motile sperm, the pregnancy rate per cycle was 67%. 75 The number of ovulations as well as recovered embryos is higher in spring and early summer than in late summer and autumn. It is generally known that the reproduction cycle of horses starts naturally when daylight is extending. The time of mating and giving birth in the horse is spring and early summer. Our results for OR as well as RE were in agreement with this facts. Hence, spring is naturally the best time to start embryo recovery in the year. The preparation of the donor mare enables a better timing of insemination as well as a better timing of transfer in the recipient mare. Application of heat- and ovulation inducing agents makes the recovery and the transfer easier to schedule, because of knowing a narrow time slot when ovulation will occur. Therefore the number of recovered embryos are higher in treated mares than in mares that got no treatment at all. Considering the running number of flushing within seasons, there is no significant difference between different flushes concerning the number of recovered embryos. This is contradictory to the result obtained from ovulation rate; number of ovulations increases with the increasing of number of flushing, so we would expect a higher number of recovered embryos. This may fail due to poorer quality of the ovulated oocytes. There was also no consistency within donor mares over the years. If a donor mare provided many embryos in one year, it was no guarantee, that she did it again next season. Generally the flushing of donor mares ranged from day 6 to day 9 after ovulation [41]. A day nine embryo, however, has grown to a size, where pregnancy rates after transfer may be decreased, potentially by damaging the embryo during handling. In this study no significant differences could be detected by flushing donor mares at day 6 to 10 after ovulation. A possible reason for the consistent performance may be the experienced veterinarians who performed the flushes. 76 Heritabilities and correlation for fertility traits in the horse were not estimated until yet. In the case of ET, it is important to consider the complexity of various traits and influencing factors; knowledge about their variance and covariance components for maternal and paternal effects is of basic interest for the genetic improvement in horse breeding and was not investigated in previous equine studies. As expected for fertility parameters heritabilities for both traits were low, although the heritability for OR was a little bit higher than for RE in the donor mare. The slightly higher heritability of the stallion on the trait RE was a little bit surprising, we imagined that the donor mares’s influence would be higher. The correlation between RE and OR showed a slight positive trend, whereas genetic correlations between maternal and paternal components show a slightly negative tendency, indicating a genetic antagonism. Although this data set is very big for the equine domain, high standard errors indicate that there may be a need for a larger data set. In cattle, genetic correlations between maternal and paternal effects for most fertility traits are reported to be negative [27]. As König et al. [26] demanded there is a need for studies revealing the physiological mechanisms for the phenomenon of slightly negative correlations between maternal and paternal effects for traits related to ET in the future not only for cattle but also for horses. A large variation in pregnancy rates was found after non surgical transfer by different authors ranging from 50 to 75 % [7, 19, 38, 69]. This is in agreement with our results showing a positive result of PD I of 66.7 % and a success rate of 51.5% regarding the living born foals. Also lower pregnancy rates were found after performing nonsurgical transfer (27% vs. 53%, [65]; 45% vs. 72%, [58]) as well as other authors reported acceptable pregnancy rates after nonsurgical transfer like 75 to 77% (66; 67). No significant differences in PD I were detected depending on the age of the recipient mare at time of transfer. This is in contrast to the influence of the age on 77 embryo recovery rate in donor mares founded in previous studies, where mares older than 16 years donate fewer numbers of embryos. Hence, the ability to become pregnant is independent of the age of the recipient mare in contrast to the production of an embryo, which depends on the age of the donor mare. Regarding the season, there is a significant influence on PD I. Higher numbers of positive PD I were achieved in spring and early summer, but generally decreased in midsummer. In spite of the fact, there is no clear trend between the years. It is generally known that the reproduction cycle of horses starts naturally when daylight is extending. Hence, spring and early summer are the best time to achieve acceptable pregnancy rates after embryo transfer. For the years 2005, 2006 and 2008 a rise of positive pregnancy determinations in late summer and autumn was found. This increase may be depending on the weaning of the foals, because mares with no foal at foot are often more likely to become pregnant. The treatment of the recipient mare differs only between recipient mares that got no treatment at all and processed mares independent of the treatment were heat inducing agents or ovulation inducing agents. Treated mares showed higher numbers of positive PD I than untreated mares. The management of the recipient mare plays an important role and therefore the timing and detection of the ovulation is of great importance for the success of embryo transfer. In treated mares this is more predictable than in untreated mares. Regarding the age of the embryo at time of transfer, best pregnancy rates were obtained when transferring Day 7 embryos or Day 8 embryos. Day 9 embryos resulted in significant lower pregnancy rates due to potential damage during the flushing and handling before transfer. This result is in agreement with Squires et al., [63] and Iuliano et al., [58], who reported reduced pregnancy rates after the transfer of large embryos. Important for the freezing of embryos (flushing at day 6 after 78 ovulation), day 6 embryos showed no significant differences regarding the pregnancy rate compared to day 7, day 8 and day 9 embryos. Especially for frozen embryos the viability and therefore acceptable pregnancy rates plays an important role, because of the forces of the freezing and thawing process. Due to individual variation of the heat length, the exact timing of ovulation in donor and recipient is, however, difficult. An approach to handle estrus synchrony of donor and recipient is the employment of induced ovulation in the recipient once an ovulation has been detected in the donor mare. The highest initial pregnancy rates of >75% at day 12 and around 65% at day 50 will be achieved when the recipient mare’s ovulation occurs within a time range of two days before to three days after the donor mare’s ovulation (19; 64). Even though some investigators found a substantially influence of estrus synchrony on the success of embryo transfer, i.e. the pregnancy rate (1; 19; 38; 62; 63), in our study no significant influence of synchrony of donor and recipient could be detected. Recipient mares ovulated in a range of zero days up to five days after the donor mare, followed by comparable pregnancy rates after embryo transfer. No variation between the recipient mares could be found, due to the failure of generating a value for the covariance parameter estimates. Comparing colts and fillies revealed in significant differences in all analyzed traits. The comparison of embryo transfer and non embryo transfer foals showed only a significant influence with respect to the length of gestation but not to withers height at birth and weight at birth. No interaction was found between the type of the foal (ET foal/Non – ET foal) and the gender. Concerning withers height at birth no significant difference was detected between ET and Non – ET foals. However, there was a significant difference regarding the gender of the foal. Colts were 0.7 cm taller than fillies, maybe due to a significant longer 79 length of gestation. Satuè et al., [59] found also a significant influence of the mare’s age on withers height at birth, agreeing with our results, showing that younger mares (≤ 6 years old) and mares, older than 16 years, give birth to smaller foals. There is a significant influence of the mare’s age with an increasing height until the age of mare of 9, 10, 11, but decreasing in mares older than 11 years old. Cilek [68] investigated Purebred Arabian foals and found a significant influence of gender of the foal on the birth weight as well. The weight at birth was 46.27 ± 0.40 kg for colts, whereas fillies weighted 45.22 ± 0.40 kg at birth. Satuè et al., [59] found younger mares (≤ 6 years) foaling offspring with little weights compared with mares older than eight years foaling heavier offspring. As reported by Elliot et al., [70] who found a significant difference in birth weights of 1.1 kg between colts and fillies, we found results of higher birth weights in colts of 1.4 kg. Likewise, Elliot et al., [70] found an increase of the weight at birth with an increase of the mare’s age up to 12 years. Compared to our results we found an increase of the weight at birth up to an age of the mare of 9 years, followed by a light decrease in weight at birth for older mares. The length of gestation is influenced by the gender of the foal as well as the type of the foal (embryo transfer/non embryo transfer). Allen et al., (60) found a mean duration of gestation of 339.0 ± 3.0 days in Thoroughbred mares. Valera et al., [61] found mean length of gestation of 336.8 ± 0.48 days in Andalusians or Spanish Purebreds and 340.3 ± 0.63 days in Arabian mares. They found that two of the main factors significantly affecting gestational length are the age of the mother and the sex of the foal. Satuè et al., [59] reported a mean gestational length of 332.4 ± 12.1 days in Carthusian broodmares, which was significantly prolonged (5.7 days) when mares give birth to colts (336.8 ± 11.0 days) rather than fillies (331.1 ± 12.8 days). In our study where we found a longer length of gestation for colts of 2 days. The range is 80 not that big as in the study above, but showed a significant longer length of gestation for colts rather than fillies as well. Satuè et al., [59] also found a 5.3 days shorter pregnancy in mares 8 to 12 years (330.8 ± 11.0 days) old in contrast to those of 13 to 17 years (336.1 ± 8.9 days). This is also in agreement with our results, that mares (>10 years old) are significantly longer in foal than younger mares. Younger mares (≤7 years old) exhibit a significantly shorter gestational length (- 4 days) than mares older than 17 years. The comparison of ET and Non – ET foals resulted in a difference of gestational length. ET foals showed a shorter length of gestation than Non – ET foals. Although there are high standard errors in the present study, it is obvious, that the season (= month of birth) shows a significant influence on length of gestation, which decreases, when the foals are born in late summer or autumn. But there is no clear trend between the years. Maybe there is a hormonal influence on the foal due to decreasing daylight, triggering the birth. There are a lot of factors that influence the procedure embryo transfer. It would be desirable, if there will be further research, especially with regard to the development of the foals, to confirm these results. REFERENCES [1] Squires, E. L.,K. J. Imel,M. F. Iuliano and R. K. Shideler. Factors affecting reproductive efficiency in an equine embryo transfer programme. J Reprod Fertil Suppl 1982;32:409-414. [2] Douglas, R. H. Some aspects of equine embryo transfer. J Reprod Fertil Suppl 1982;32:405-408. [7] Vogelsang, S. G.,K. R. Bondioliand J. M. Massey. Commercial application of equine embryo transfer. Equine Veterinary Journal 1985;17:89-91. 81 [15] Ball, B. A.,T. V. Little,J. A. Weberand G. L. Woods. Survival of day-4 embryos from young, normal mares and aged, subfertile mares after transfer to normal recipient mares. Journal of Reproduction and Fertility 1989;85:187-194. [19] McKinnon, A. O., Squires, E. L., Voss, J. L. and Cook, V. M.; Equine embryo transfer. Compendium on Continuing Education for the Practicing Veterinarian 1988; 10: 343-355. [21] Squires, E. L.,M. G. McClain,O. J. Gintherand A. O. McKinnon. Spontaneous multiple ovulation in the mare and its effect on the incidence of twin embryo collections. Theriogenology 1987;28:609-613. [24] Vogelsang, S. G.and M. M. Vogelsang. Influence of donor parity and age on the success of commercial equine embryo transfer. Equine Veterinary Journal 1989;21:71-72. [26] Koenig, S.,F. Bosselmann,U. U. von Borsteland H. Simianer. 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G.; Relationship between the fertility of fresh and frozen stallion semen and semen quality. Journal of reproduction and fertility. Supplement 1991; 44: 107-14. [55] Dobrinski, I., Thomas, P. G. A. and Ball, B. A.; Cryopreservation reduces the ability of equine spermatozoa to attach to oviductal epithelial cells and zonae pellucidae in vitro. Journal of Andrology 1995; 16: 536-542. [56] Ellington, J. E., Samper, J. C. and Wright, W. R. (2000). Stallion sperm interactions with oviduct cells in vitro as an indicator of field fertility. 83 Proceedings the 14th International Congress Animal Reproduction, Stockholm, Sweden. [57] Brinsko, S. P.; Insemination doses: How low can we go? Theriogenology 2006; 66: 543-550. [58] Iuliano, M. F., Squires, E. L. and Cook, V. M.; Effect of age of equine embryos and method of transfer on pregnancy rate. Journal of Animal Science 1985; Vol. 60, No. 1: 258-263. [59] Satué, K., Felipe, M., Mota, J. and Muñoz, A.; Gestational length in Carthusian broodmares: effects of breeding season, foal gender, age of mare, year of parturition, parity and sire. Polish Journal of Veterinary Science 2011; Vol. 14, No. 2: 173-180. [60] Allen, W. R., Wilsher, S., Turnbull, C., Stewart, F., Ousey, J., Rossdale, P. D. and Fowden, A. L.; Influence of maternal size on placental, fetal and postnatal growth in the horse. I. Development in utero. Reproduction 2002; 123: 445453. [61] Valera, M., Blesa, F., Dos Santos, R. and Molina, A.; Genetic study of gestation length in andalusian and arabian mares. Animal Reproduction Science 2006; Vol. 95, Issue 1-2: 75-96. [62] Squires, E. L., Garcia, R. H. and Ginther, O.J.; Factors affecting success of equine embryo transfer. Equine Vet. J. 1985; 3(Suppl.): 92-95. [63] Squires, E. L., Cook, V. M. and Voss, J. L.; Collection and transfer of equine embryos. Colorado State University Animal Reproduction and Biotechnology Lab. Bulletin 1985; 1: 16-25. [64] Carney, N. J., Squires, E. L., Cook, V. M., Seidel Jr., G. E. and Jasko, D. J.; Comparison of pregnancy rates from transfer of fresh versus cooled, transported equine embryos. Theriogenology 1991; Vol. 36, No. 1: 23-32. [65] Imel, K. J., Squires, E. L., Elsden, R. P., Shideler, R. K.; Collection and transfer of equine embryos. J. Amer. Vet. Med. Assoc. 1981; 179-987. [66] Hinrichs, K. and Kenney, R. M.; Effect of timing of progesterone administration on pregnancy rate after embryo transfer in ovariectomized mares. J. Reprod. Fertil. 1987; Suppl. 35: 439-443. [67] Wilson, J. M., Rowley, M. B., Rowley, W. K., Smith, H. A., Webb, R. L. and Tolleson, D. R.; Successful non-surgical transfer of equine embryos to postpartum mares. Theriogenology 1987; 27: 295. (Abstr.) 84 [68] Cilek, S.; The survey of reproductive success in Arabian horse breeding from 1976-2007 at Anadolu State farm in Turkey. J. Anim. Vet. Adv. 2009; 8: 389396. [69] Riera, FL., and McDonough, J.; Commercial embryo transfer in polo ponies in Argentina. Equine Vet. J. 1993; 15(Suppl): 116-119. [70] Elliot, C., Morten, J., Chopin, J.; Factors affecting foal birth weight in Thoroughbred horses. Theriogenology 2009; 71: 683-689. 85 86 General Summary Equine embryo transfer includes the insemination and the following embryo recovery of the donor mare as well as the transfer into the recipient mare. The recipient takes over the pregnancy and the raising of the foal. Due to numerous process steps, there are several influencing factors, concerning the success of embryo transfer in a positive or negative way. In our study we investigated possible influencing factors, affecting the traits “number of ovulations” and “number of recovered embryos” in the donor mare. The influencing factors on the pregnancy rates after transfer in the recipient mare were investigated as well as the number of living born foals. Embryo transfer foals were compared to non embryo transfer foals regarding the length of gestation, withers height at birth and weight at birth. Also a comparison between colts and fillies was done, with respect to this traits. The influence of the age of the (recipient) mare with regard to the same traits was also investigated. Additionally, the influence of the season was tested for the trait “length of gestation”. Furthermore heritabilities and correlations for fertility parameters “number of ovulations” and “number of recovered embryos” were estimated. In Chapter One the following influencing factors with regard to the probability for a successful flushing (recovery of at least one embryo) were investigated: age of the donor mare at time of flushing, the month, where flushing is performed, the preparation of the donor mare prior to the flushing (application of heat-/ovulation inducing agents) as well as the running number of flushing within donor mare and season. No significant differences could be detected at an donor mare’s age between 2 and 15 years. Only donor mares (>15 years old) showed a significant lower number of recovered embryos. 87 With respect to the season (month of flushing) the highest probability for a successful flushing was found in spring and early summer. The success rate decreased, if the flushing was performed in late summer and autumn. Regarding the preparation of the donor mare, a significant higher number of recovered embryos were obtained after application of ovulation inducing agents, with or without a prior application of heat inducing agents. The running number of flushes within donor mare and season showed no significant influence on the embryo recovery rate. As expected for fertility parameters, heritabilities the for embryo recovery rate were low (3.7% for the donor mare and 4.5% for the service stallion). The service stallion showed a slightly higher influence on the success of flushing than the donor mare. Additionally following influencing factors with regard to the probability of a successful flushing were investigated in Chapter Two: type of semen (fresh or frozen semen), motility of the spermatozoa as well as the number of spermatozoa and the breeding status of the donor mare. The use of fresh semen resulted in a significant higher number of recovered embryos, than insemination with frozen semen. Furthermore utilization of spermatozoa with a motility of >65% resulted in significant higher embryo recovery rates as well. The number of spermatozoa as well as the breeding status of the donor mare showed no significant influence on the success of flushing. Chapter Three contains the investigation of the following influencing factors with respect to positive pregnancy determination around 9 days after transfer in the recipient mare. The age of the recipient mare at time of transfer showed no significant influence on the pregnancy rate. Despite of some differences between the years, spring was the most suitable time to obtain a positive pregnancy result after embryo transfer. 88 The preparation of the recipient mare showed significant higher pregnancy rates in treated recipients, than in recipients that got no treatment at all. The age of the embryo at time of transfer resulted in significant higher pregnancy rates for 7 and 8 day old embryos, than for those who were 9 days old. The comparison of embryo transfer to non embryo transfer foals resulted only in a significant shorter length of gestation (0.7 days) for embryo transfer foals. Withers height at birth and weight at birth showed no significant differences between both groups. Also there was no interaction between the “type” of the foal (embryo transfer/non embryo transfer) and the gender of the foal. However, the gender of the foal showed a significant influence on length of gestation, withers height at birth as well as weight at birth. Length of gestation was in colts 2 days longer, colts were 0.7 cm taller and 1.4 kg heavier at birth. The age of the mares at time of birth resulted in the following significant differences: Mares (>16 years old) had a longer pregnancy than younger mares. Mares (<6years old and >16 years old) gave birth to smaller foals (lower withers height at birth). Furthermore the foals of mares (<8 years old) showed lower weight at birth. Due to low heritabilities for fertility parameters on the one hand and breeding goals for sport horses (improvement of dressage and/or jumping ability) on the other hand, the practical use for breeding is limited. The improvement of the management of equine embryo transfer should be the focus of future action . 89 90 Zusammenfassung Das Verfahren Embryo Transfer beim Pferd setzt sich aus der Besamung und der folgenden Gewinnung der Embryonen aus der Spenderstute sowie dem anschließenden Transfer auf die Empfängerstute zusammen, die dann die Trächtigkeit und die Aufzucht des Fohlens übernimmt. Aufgrund der zahlreichen Verfahrensschritte gibt es viele Faktoren, die den Erfolg des Embryo Transfer in positiver oder negativer Weise beeinflussen. In dieser Arbeit wurden mögliche Faktoren untersucht, die die Merkmale „Anzahl der Ovulationen“ und Anzahl der gewonnen Embryonen“ in der Spenderstute beeinflussen können. Für die Empfängerstuten wurden sowohl die Trächtigkeitswahrscheinlichkeiten nach dem Transfer, inklusive der einwirkenden Faktoren ermittelt als auch die Anzahl lebendgeborener Fohlen. Die Fohlen aus Embryo Transfer wurden mit den Fohlen, die von ihren genetischen Müttern selbst ausgetragen wurden, hinsichtlich Trächtigkeitsdauer, Widerristhöhe bei Geburt und Geburtsgewicht verglichen. Ebenso fand ein Vergleich in diesen Merkmalen zwischen Hengst- und Stutfohlen statt. Der Einfluss des Alters der (Empfänger-) Stute auf diese Merkmale wurde gleichfalls untersucht. Das Merkmal Trächtigkeitsdauer wurde zusätzlich noch auf den Einfluss der Saison untersucht. Weiterhin wurden Heritabilitäten und Korrelationen für die Fruchtbarkeitsmerkmale „Anzahl der Ovulationen“ und „Anzahl der gewonnenen Embryonen“ geschätzt. In Kapitel Eins wurden folgende signifikante Einflussfaktoren auf die Wahrscheinlichkeit einer erfolgreichen Spülung (Gewinnung von mindestens einem Embryo) untersucht: Alter der Spenderstute zum Zeitpunkt der Spülung, der Monat, in dem die Spülung durchgeführt wurde, die Vorbereitung der Spenderstute auf die Spülung (Gabe von rosse- bzw. ovulationsindizierenden Wirkstoffen) sowie die 91 Anzahl der Spülungen einer Spenderstute innerhalb einer Saison. Keine signifikanten Unterschiede in der Anzahl der gewonnenen Embryonen konnten bei Spenderstuten im Alter von 2 bis 15 Jahren festgestellt werden. Lediglich Spenderstuten, die älter als 15 Jahre waren, zeigten eine signifikant geringe Anzahl an gewonnenen Embryonen. Die höchste Wahrscheinlichkeit einer erfolgreichen Spülung wurde in den Frühlings- und frühen Sommermonaten festgestellt. Die Erfolgsrate sank, wenn die Spülungen im späten Sommer und Herbst durchgeführt wurden. Die Vorbereitung der Spenderstute auf die Spülung zeigte, dass Spenderstuten, die einen ovulationsinduzierenden rosseinduzierenden Wirkstoff, Wirkstoffs, mit oder appliziert ohne vorheriger bekamen Gabe signifikant einen höhere Wahrscheinlichkeiten einer erfolgreichen Spülung aufwiesen, als Spenderstuten, die nur rossestimuliert wurden bzw. gar nicht stimuliert wurden. Die Anzahl der durchgeführten Spülungen pro Stute/Jahr zeigte keinen signifikanten Einfluss auf die Embryonengewinnungsrate. Wie für Fruchtbarkeitsparameter zu erwarten war, lag die Heritabilität für das Merkmal „Anzahl gewonnener Embryonen“ in einem niedrigen Bereich (3,7% für die Spenderstute und 4,5% für den Besamungshengst). Der Besamungshengst zeigte somit einen etwas höheren Einfluss auf den Erfolg der Spülung als die Spenderstute. Zusätzlich wurden in Kapitel Zwei folgende Einflussfaktoren auf die Wahrscheinlichkeit einer erfolgreichen Spülung untersucht: Art des zur Besamung der Spenderstute eingesetzten Spermas (Frisch- /Tiefgefriersperma), die Vorwärtsbeweglichkeit der Spermien sowie die Anzahl der Spermien und der Zuchtstatus der Spenderstute. Der Einsatz von Frischsperma führte zu einer signifikant höheren Anzahl gewonnener Embryonen, als der Gebrauch von Tiefgefriersperma. Weiterhin ergab der Einsatz von Sperma mit einer Motilität von >65% eine signifikant höhere Wahrscheinlichkeit für eine erfolgreiche Spülung. Keinen signifikanten Einfluss auf die Embryonengewinnungsrate zeigten 92 die Anzahl der Spermien und der Zuchtstatus der Spenderstute. In Kapitel Drei wurden folgende Einflussfaktoren auf die Wahrscheinlichkeit einer erfolgreichen Trächtigkeit der Empfängerstute untersucht: Das Alter der Empfängerstute zum Zeitpunkt des Transfers zeigte keinen signifikanten Einfluss auf die Trächtigkeitsrate. Trotz gewissen Unterschieden zwischen den Jahren, zeigten sich, die Frühlingsmonate am geeignetsten für eine erfolgreiche Trächtigkeit nach Embryo Transfer. In der Vorbereitung der Empfängerstute auf den Transfer zeigte sich, dass Empfängerstuten, die rosse- und/oder ovulationsstimuliert wurden, signifikant höhere Trächtigkeitsraten aufwiesen, im Gegensatz zu denen, die nicht stimuliert wurden. Das Alter des Embryos zum Zeitpunkt des Transfers zeigte signifikant höhere Trächtigkeitsraten bei der Nutzung von sieben bzw. acht Tage alten Embryonen, als bei Embryonen, die neun Tage alt waren. Der Vergleich zwischen „Embryo Transfer Fohlen“ und „Nicht Embryo Transfer Fohlen“ ergab eine etwas kürzere Trächtigkeitsdauer (0,7 Tage) für die Embryo Transfer Fohlen. Das Geburtsgewicht und die Größe bei der Geburt zeigten keine signifikanten Unterschiede zwischen den beiden Gruppen. Ebenfalls konnte keine Interaktion zwischen dem „Typ“ des Fohlens (Embryo Transfer/Nicht Embryo Transfer) und dem Geschlecht des Fohlens festgestellt werden. Der Einfluss des Geschlechts des Fohlens auf die Merkmale Trächtigkeitsdauer, Größe bei Geburt und Geburtsgewicht war hingegen signifikant. Die Trächtigkeitsdauer war bei Hengstfohlen im Mittel 2 Tage länger, die Hengstfohlen waren bei Geburt 0,7 cm größer und 1,4 kg schwerer, als die Stutfohlen. Das Alter der Stuten zum Zeitpunkt der Geburt zeigte folgende signifikante Unterschiede: Stuten (>16 Jahre alt) waren länger tragend als jüngere Stuten. Stuten (<6 Jahre alt und >16 Jahre alt) bekamen kleinere Fohlen (geringer Widerristhöhe bei Geburt). Weiterhin bekamen Stuten (<8 Jahre alt) Fohlen mit geringeren Geburtsgewichten. 93 Aufgrund der geringen Heritabilität für Fruchtbarkeitsmerkmale und der Zuchtziele für Sportpferde (Verbesserung der Dressur- und /oder Springleistung) ist der praktische, züchterische Nutzen begrenzt. Die Optimierung des Embryo Transfers sollte im Management des Verfahrens liegen. 94 Danksagung An dieser Stelle sollen die Personen erwähnt werden, die mich im Rahmen meiner Promotion begleitet haben und zum Gelingen dieser Arbeit beigetragen haben. Mein Dank gilt Herrn Prof. Dr. Georg Thaller für die Überlassung des sehr interessanten Dissertationsthemas, die wissenschaftliche Unterstützung und die Chance meine Ergebnisse auf nationalen und internationalen Workshops, Tagungen und Kongressen zu präsentieren. Mein besonderer Dank gilt Herrn Paul Schockemöhle für die Bereitstellung der Daten dieser Arbeit, die kooperative Zusammenarbeit und die Möglichkeit ein dreiviertel Jahr lang den Alltag auf Gestüt Lewitz erleben zu können. Weiterhin bin ich Herrn Prof. Dr. Dr. h.c. mult. Ernst Kalm sehr verbunden für die Begleitung durch die Zeit der Promotion und die Möglichkeit jederzeit „hippologische Fragen“ stellen zu können. Zu größtem Dank bin ich Dr. Eckhard Stamer verpflichtet, der wirklich jederzeit für Fragen zur Verfügung stand und immer eine Lösung für statistische Probleme parat hatte. Seine motivierende Art war stets ein großes Vorbild für mich. Herrn Dr. Jens Tetens sei herzlich gedankt für sein „sehr spontanes Einspringen“ beim Korrigieren, für seine guten Englischkenntnisse, die zum Gelingen der Arbeit wesentlich beigetragen haben und sein Einsatz für das Veröffentlichen der Artikel in den Fachjournals. Für die finanzielle Unterstützung im Rahmen eines Stipendiums danke ich sehr herzlich der H. Wilhelm Schaumann Stiftung. Herrn Dr. Marc Lämmer, Dr. Roberto Sanchez und „Gyn-Team“, sowie dem ganzen Team des Gestütes Lewitz danke ich sehr für die tolle Zeit und die herzliche Aufnahme. 95 Ich danke meinen „Wintermitbewohnerinnen“ Karina, Wiebke und Kristina für die grandiose Zeit, die Planung der „Worldtour“, die zahlreichen Kinobesuche und die tolle Freundschaft, die sich daraus entwickelt hat. Ganz besonders bedanken möchte ich mich bei meinen beiden Bürokollegen Gesche und Tobias. Es war eine phantastische Zeit mit Euch, die ich wirklich vermisse. Vielen Dank an alle Mitglieder des Instituts für Tierzucht und Tierhaltung; ich habe mich im Haus immer sehr wohl gefühlt und es hat mir großen Spaß gemacht dort zu arbeiten. Der Campus Suite danke ich für das „rund-um-die-Uhr“ Bereitstellen von Kaffespezialitäten und leckeren Snacks. Für das „Cat and Cavia Sitting“ möchte ich mich ganz herzlich bei Marita und Christiane bedanken; ohne Euch wäre das alles nicht so unkompliziert möglich gewesen. Meiner Familie gehört der größte Dank, insbesondere meinen Eltern, die mich immer selbstverständlich unterstützt haben und so maßgeblich an dem Gelingen der Arbeit beteiligt waren. Zum Schluss möchte ich mich noch Maren, Christian, Claudia, Thekla-Karina, Simone, Steffi, Kristina, Jens und Marco für eine tolle, sehr offene Freundschaft bedanken. 96 GREFFENER STRASSE 1 • 33775 VERSMOLD MOBIL 0173/2888505 • E-MAIL [email protected] HENRIK VON DER AHE PERSÖNLICHE INFORMATIONEN Familienstand: ledig Nationalität: deutsch Alter: 33 Geburtsort: Münster Eltern: Horst von der Ahe (Apotheker) Marianne von der Ahe (Pharmazeutisch-technische Assistentin) AUSBILDUNG 1985 bis 1989 Kreuzschule (Grundschule), Münster 1989 bis 1998 Pascalgymnasium, Münster 1998 bis 1999 Zivildienst Raphaelsklinik GmbH, Münster 1999 bis 2001 Ausbildung zum Pferdewirt Schwerpunkt Zucht und Haltung, Hessisches Landgestüt Dillenburg 2001 bis 2002 Studium der Pharmazie an der CAU Kiel (2 Semester) 2002 bis 2008 Studium der Agrarwissenschaften an der CAU Kiel, 2007 Abschluss Bachelor of Science 2008 Abschluss Master of Science 2008 bis 2011 Promotion am Institut für Tierzucht und Tierhaltung der CAU Kiel 2011 bis heute Nachwuchsführungskraft bei Deutsche Reiterliche Vereinigung e.V. (FN), Warendorf 97