Reproductive Parameters of Mangalarga Marchador Mares in a Commercial Embryo Transfer Programme

Reprod Dom Anim 46, 261–267 (2011); doi: 10.1111/j.1439-0531.2010.01656.x ISSN 0936-6768

Reproductive Parameters of Mangalarga Marchador Mares in a Commercial Embryo Transfer Programme E de P Lopes1, JB Siqueira2, RO Pinho3, JD Guimara˜es4, AN Rocha1, GR de Carvalho4 and CAA Torres4 1 Ms Autonomous Veterinary; 2Faculdade de Medicina Veterina´ria e Zootecnia ⁄ UNESP – Botucatu, SP, Brasil; 3Departamento de Veterina´ria, Universidade Federal de Vic¸osa, MG, Brasil; 4Departamento de Zootecnia, Universidade Federal de Vic¸osa, MG, Brasil

Contents The objective of this study is to evaluate the reproductive efficiency in donors and recipient Mangalarga Marchador mares in commercial programmes of embryo transfer (ET) and the effects of some reproductive characteristics and ET methodology on conception rates in the recipient mares. A total of 1140 flushing procedures were performed and 830 embryos (72.8%) were recovered. There were no differences between the rates of embryonic recovery in the different breeding seasons (p > 0.05) and 92.8% of the recovered embryos were 8–9 days old. There was no difference in the embryonic recovery regarding the collection order from the first to the ninth embryo collection along the breeding season, as well as among mares inseminated during the foal heat or subsequent cycles (p > 0.05). Pregnancy rates observed in the total period of all reproductive seasons at 15, 30, 45 and 60 days of pregnancy were 73.4, 69.9, 66.7 and 64.5%, respectively. Differences in pregnancy rate and early embryonic loss rates were not observed between embryos transferred immediately after collection (66.8% and 13.5%) and embryos transported at room temperature for periods of 0.05). Pregnancy rates were higher when the interval between ovulations of donor and recipient mares remained between )3 and )2 days (p < 0.05), and the lowest rates were observed for intervals of )6 days (p < 0.05) with intermediary values for intervals of )1, 0 and +1 (p > 0.05). Embryonic loss rates, however, did not differ between intervals of ovulation’s synchronism between donor and recipient mares (p > 0.05). This flexibilization in the ovulatory synchronism between donor and recipient mares optimizes the use of recipient mares, thus reducing costs and facilitating management of horse breeding farms.

Introduction The demand for the development of techniques for assisted reproduction in equine production has recently greatly increased. Although the use of embryo transfer (ET) has increased over the past two decades, its numbers have fluctuated along with the financial health of the equine industry. Currently, the high cost of ET has dictated that only genetically superior animals are used as donors (Squires et al. 1999). Some singular biological characteristics, as well as technical problems, have limited its widespread use in equine species, when compared to bovine species (Squires et al. 1999). The major candidates for the ET programme are older mares with poor reproductive history that cannot produce a foal by natural breeding or artificial insemination, and mares that are in competition (Lopes 2002a; Squires et al. 2003), although embryonic loss rates in older mares are higher (Squires et al. 2003) and fertility decreases with the mare’s age (Merkt et al. 2000).

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The success of an ET programme is closely related to the rate of embryo recovery from donor mares. Data of five years in a Mangalarga Marchador horse breeding farm, summarizing 658 flushings, resulted in 63.4% embryo recovery rate (Jacob et al. 2002). Several factors affect the embryo recovery; among them were the number of ovulation (Squires et al. 1987; Carmo et al. 2003); the day of embryo collection (Fleury and Alvarenga 1999; Squires et al. 1999); the donor mare (Carnevale and Ginther 1992); and the quality of semen (Amann and Pickett 1987). After two decades of research and development of the art of ET in equine species, it is not uncommon to note now-a-days, pregnancy rates after ET comparable to those obtained by oestrous cycle with the use of natural breeding or artificial insemination. Two studies in developed programmes for ET with Mangalarga Marchador mares reported pregnancy rates of 70.3% (270 ⁄ 384) (Jacob et al. 2002) and 72.5% (95 ⁄ 131) (Gomes et al. 2004). The pregnancy rate is influenced by embryo age (Squires et al. 1999, 2003; Carnevale et al. 2000; Fleury et al. 2002), site of embryo deposition (Carvalho 2000), transfer method (Fleury and Alvarenga 1999; Silva 2003; Gomes et al. 2004; Pessoˆa et al. 2004), recipient mare (Jacob et al. 2002; Lopes 2002b; Squires et al. 2003), embryo quality (Squires 1993), refrigeration and transport of the embryo (Fleury et al. 2002; Squires et al. 2003), interval between delivery and the first heat, foaling interval, and the use of foal heat (Camillo et al. 1997; Carvalho 2000; Malschitzky et al. 2002). This study deals with the characterization and evaluation of the reproductive efficiency of donor and recipient Mangalarga Marchador mares in commercial programmes for ET, and the verification of the relationship between reproductive and productive features or ET techniques and pregnancy rates in recipient mares.

Materials and methods The data for the research were obtained from four breeding farms, and six breeding seasons, during the period from 1998 to 2004. Each breeding season lasts from August to May. Breeding farm 1 was located in Resende-RJ, at the latitude of 2228¢08¢¢S and longitude of 4426¢49¢¢W (season 1998 ⁄ 99); breeding farm 2 was located in Jequitiba´-MG, at 1914¢08¢¢S and 4401¢40¢¢W (seasons 1999 ⁄ 00 and 2000 ⁄ 01); breeding farm 3 was located in Ponte Nova-MG, at 2024¢59¢¢S and 4254¢31¢¢W (seasons 2001 ⁄ 02 and 2002 ⁄ 03); and

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breeding farm 4 was located in Esmeraldas-MG, at 1945¢45¢¢S and 4418¢50¢¢W (season 2003 ⁄ 04). Two hundred and nine donor mares were used, aging from five to 20 years and none of them was competing. They were kept in grassy pastures (Cynodon spp., coastcross), except for those animals that were in preparation for competitions, which were held in stalls with water and mineralized salt ad libitum, and fed with diced forage grass or hay, and concentrated dry food twice daily, usually when they were taken to the pen, for reproductive evaluation. The mares were examined at intervals of 2 or 3 days, until a 30-mm follicle was detected, when they began to be examined daily. During palpations, ovarian structures (follicles or corpus luteum), uterine tonus and degree of cervix opening were reported. The ultrasound scans of the ovaries and uterus were performed with either a Vet 200 (Pie Medical, Maastricht, Netherland) or a SD 500 (ALOKA, Tokyo, Japan) with a 5-MHz transrectal transducer. The donor mares were mated or inseminated every 48 h since the detection of a ‡35-mm follicle until ovulation was detected. Semen was aspirated into a 20-ml syringe attached to an insemination pipette for mares (Provar ⁄ Sa˜o Paulo, Brazil) and deposited into the uterine body or uterine horn ipsilateral to the ovary that contained the pre-ovulatory follicle. When stallions were kept in the same farm of the donor mares, for artificial inseminations fresh semen diluted in skim milkbased extender was used; otherwise semen was diluted in the same extender, refrigerated at 15C, and utilized for up to 12 h. Embryos were collected 7–9 days after ovulation, and only grade 1 and 2 embryos (Carvalho et al. 2001a) were transferred. Approximately one to three litres of Ringer’s lactate solution was used for uterine flushings at 35C, as described by Alvarenga et al. (1993). After the observation of the embryo in the filter, the rest of the solution in the uterus was siphoned off and the collection was concluded. If no embryo was observed at the filter, the procedure was repeated twice. At the end of the collection, the donor mare received an intramuscular application of prostaglandin-F2a (7.5 mg, Lutalyse ⁄ Pfizer, NY, USA), for induction of a new cycle. After flushing, fluid was screened in stereomicroscope, and the embryos were transferred immediately to a plate containing embryo maintaining medium (Emcare ⁄ ICP, Auckland, New Zealand). The embryos were washed 4–5 times in the medium before being packed for transfer. When the flushing fluid was considered dirty or when cellular debris were adhered to the embryo, they were transferred to another plate with medium and the washing procedure was repeated. Two types of devices were used to transfer, according to the size of the recovered embryos. Smaller embryos ( 0.05).

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8 days

7 days

9 days

80 70.2

67.3

70

75.7

66.7

69.4

60

58.3

50 41.7

% 40 30

25

23.4

10

23.8 17.5

20

13.1 7.7

17.8 9.5

6.4

6.5 0

0

Fig. 1. Percentage of embryos 7, 8, and 9 days old, recovered in the different analysed breeding seasons

1998/1999

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Total

Total collections % (n) 21.3 18.5 15.0 12.4 10.2 7.6 5.3 3.3 2.7 1.7 1.0 0.6 0.3 0.2 1140

(243) (211) (171) (141) (116) (87) (60) (38) (31) (19) (11) (7) (3) (2) (100.0)

Negative Collections % (n)

1 Embryo recovered % (n)

2 Embryos recovered % (n)

3 Embryos recovered % (n)

28.0 28.0 32.8 36.9 31.9 31.0 30.0 31.6 32.3 31.6 27.3 28.6 33.3 – 30.7

68.7 67.3 65.5 59.6 64.7 65.5 66.7 65.8 58.1 68.4 63.6 71.4 66.7 100.0 65.7

2.9 (7) 4.7 (10) 2.3 (4) 3.5 (5) 3.4 (4) 3.4 (3) 3.3 (2) 2.6 (1) 9.7 (3) – 9.1 (1) – – – 351.0 (40)

0.4 (1) – – – – – – – – – – – – – 0.1 (1)

(68) (59) (55) (52) (37) (27) (18) (12) (10) (6) (3) (2) (1) (350)

2000/2001

2001/2002

2002/2003

2003/2004

Breeding season

Table 2. Embryo recovery rate* according to the order of the oestral cycle in the breeding season in an embryo transfer programme in Mangalarga Marchador mares Oestrual cycle ordera

1999/2000

(167) (142) (112) (84) (75) (57) (40) (25) (18) (13) (7) (5) (2) (2) (749)

*p > 0.05 in chi-square test (v2GL1 = 3,84). Collection orders from 10 to 14 were not considered because of the number of repetitions. a

Table 3. Embryo recovery rates in Mangalarga Marchador mares, inseminated in the foal heat or other heats Type of heat

Collections

Recovered embryos (%)

Foal heat Other heats Total

14 1126 1140

11 (78.6)a 819 (72.7)a 830 (72.8)

Values followed by the same letters in the same column do not differ (v2GL1 = 3.84; p > 0.05).

same donor in the same breeding season (p > 0.05; Table 2). Embryo recovery rates between mares inseminated during the foal heat and other heats were compared, and under the conditions of this study, no difference was detected between the two groups (p > 0.05; Table 3). Table 4 shows the pregnancy rates and early embryonic loss at days 15, 30, 45, and 60 after ovulation of the donor, respectively. During the period from 15 to 60 days of gestation, differences in pregnancy rate between seasons were observed.  2010 Blackwell Verlag GmbH

The pregnancy and early embryonic loss rates for embryos transferred immediately after collection, compared to those collected and transported at environmental temperature for periods of 0.05; Table 5). The intervals of ovulation synchrony between donor and recipient mares used in this study are summarized in Table 6. This wide range between recipient and donor is because in Brazil the recipients are required to be registered and of the same breed as the donor mares, so it is difficult to obtain a satisfactory number of recipients in a commercial ET programme. From a total of 792 mares with data evaluated for this feature, 97.23% (n = 770) were between )6 and +1 days intervals in relation to the donor ovulation. Because of the low number of recipients (n = 22) used with the synchrony of +3 (n = 3), +2 (n = 9), )7 (n = 8), and )8 (n = 2), these data were not used in the analysis of the synchrony of ovulation between donors and recipients. However, those mares returned a rate of 54% of pregnancy (12 ⁄ 22), and there was no loss of pregnancy in this group of recipients. There was no difference in early embryonic loss rate between different intervals of synchrony of ovulation between donor and recipient (+1 to )6; p > 0.05).

Discussion The total embryo recovery rate during six breeding seasons was 72.8% (Table 1), and it was higher than the 53% reported by Reilas et al. (2000), in 15 mares inseminated 13 h after the first postpartum ovulation, and by Jacob et al. (2002) with 63.4% in the Mangalarga Marchador mares during three breeding seasons, and other studies also on a large scale, but with other breeds (Fleury et al. 1989; Fleury and Alvarenga 1999). In some cases, such as that of Fleury et al. (1989), the difference can be explained by increased age of donors, which significantly affects the embryonic recovery, as mentioned by Squires et al. (1999), or to the use of a large number of stallions with different fertilities (Amann and Pickett 1987), which is a very common practice in large-scale commercial programmes. On the other hand, in Mangalarga Marchador breeding farm only stallions of high fertility are selected as sires, and the present study used mainly these resident stallions.

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Table 4. Pregnancy rates at 15, 30, 45, and 60 days and early embryonic loss (EEL) at 30, 45, and 60 days after-ovulation of the donor in Mangalarga Marchador recipient mares belonging to embryo transfer programmes, according to the farms and breeding seasons Breeding farm 1 2 2 3 3 4 Total

Season

Transfers

1998 ⁄ 1999 1999 ⁄ 2000 2000 ⁄ 2001 2001 ⁄ 2002 2002 ⁄ 2003 2003 ⁄ 2004 –

79 188 171 139 81 172 830

% Pregnancy 15 days 67.1 71.8 71.9 65.5 80.2 82.6 73.4

(53)a (135)a (123)a (91)a (65)ac (142)bc (609)

% Pregnancy 30 days 63.4 69.7 67.2 62.6 80.3 76.7 69.9

(50)bc (131)abc (115)bc (87)c (65)a (132)ab (580)

% EEL 30 days 5.8 3.0 6.5 4.4 0.0 7.0 4.8

(3 ⁄ 53)a (4 ⁄ 135)a (8 ⁄ 123)a (4 ⁄ 91)a (0 ⁄ 65)a (10 ⁄ 142)a (29 ⁄ 609)

% Pregnancy 45 days 62.0 67.5 64.3 57.6 79.0 72.1 66.7

(49)bc (127)ab (110)bc (80)c (64)a (124)ab (554)

% EEL 45 days 7.6 5.9 10.6 12.2 1.5 12.7 9.0

(4 ⁄ 53)a (8 ⁄ 135)a (13 ⁄ 123)a (11 ⁄ 91)a (1 ⁄ 65)a (18 ⁄ 142)a (55 ⁄ 609)

% Pregnancy 60 days 59.5 65.4 62.6 54.7 76.5 69.8 64.5

(47)bc (123)abc (107)bc (76)c (62)a (120)ab (535)

% EEL 60 days 11.3 8.9 13.0 16.5 4.6 15.5 12.2

(6 ⁄ 53)a (12 ⁄ 135)a (16 ⁄ 123)a (15 ⁄ 91)a (3 ⁄ 65)a (22 ⁄ 142)a (74 ⁄ 609)

Values followed by the same letters in the same column do differ (v2GL1 = 3.84; p < 0.05). Numbers within () indicates number of pregnant mares; ( ⁄ ) indicates number of mares that aborted in relation to the total number of pregnant mares at day 15.

Table 5. Pregnancy rates after 60 days post-ovulation of the donor and early embryonic loss (EEL) in Mangalarga Marchador recipients after transfer of embryos carried or transferred immediately after collection Type of embryo

Number

Pregnancy rate n (%)

EEL n (%)

Transported Not transported Total

232 572 804

155 (66.8)a 360 (62.9)a 515 (64.0)

21 (13.5)a 52 (14.4)a 73 (14.2)a

Values followed by the same letters in the same column do not differ (v2GL1 = 3.84; p > 0.05).

Table 6. Reproductive status (pregnancy, embryonic death) in Mangalarga Marchador mares according to the ovulation synchrony between donors and recipients

Synchrony )8* )7* )6 )5 )4 )3 )2 )1 0 +1 +2* +3* Total

Pregnant (%) 100 62.5 38.5 69.2 65.9 75.9 70.6 59.4 55.9 57.6 33.3 66.7 64.9

(2) (5) (10)a (36)b (60)b (110)bc (120)bce (85)bdf (47)adf (34)adf (3) (2) (514)

Not pregnant (%) 0 37.5 53.8 25.0 23.1 19.3 18.8 28.0 34.5 35.6 66.7 33.3 26.3

(0) (3) (14) (13) (21) (28) (32) (40) (29) (21) (6) (1) (208)

Loss of Pregnancy (%) 0 0 7.7 5.8 11.0 4.8 10.6 12.6 9.5 6.8 0 0 8.8

(0) (0) (2)a (3)a (10)a (7)a (18)a (18)a (8)a (4)a (0) (0) (70)

N Mares 0.2 1.0 3.3 6.6 11.5 18.3 21.5 18.1 10.6 7.4 1.1 0.4 100.0

(2) (8) (26) (52) (91) (145) (170) (143) (84) (59) (9) (3) (792)

Values followed by different letters in the same column differ (v2GL1 = 3.84; p < 0.05). Numbers within () inducates number of mares. *As a function of the number of animals, these data were not used in analysis of the synchrony of ovulation between donors and recipients.

So, the use of stallions and donors of good fertility in different horse breeding farms and the conduction of programmes by the same professional most likely contributed to the similarity in embryo recovery results at different places and seasons. Fleury and Alvarenga (1999) concluded that with the appropriate handling and transfer techniques, the pregnancy rate resulting from 8 or 9 day old embryos are similar (74.7% and 76.5%, respectively) to those obtained from 7 days old embryos (74.5%). According to those authors, the embryo collection on the eighth day after ovulation tended to increase embryo recovery

rates (58%; p = 0.07) when compared to 7 and 9 days (49.3% and 54.5%, respectively). Carvalho et al. (2001b), in a study involving collections held on the seventh (n = 23) and on the eighth day (n = 90) after ovulation, observed embryonic recovery rates of 30.4% and 57.8%, respectively, indicating better recovery also on the eighth day (p < 0.05). Concerning the percentage of embryos with 7, 8 and 9 days of age, recovered during the different breeding seasons (Fig. 1), the range of 8–9 days of age, previously adopted by the programme, is justified by the fact that at that age, the embryos are easily visualized in the collector filter, which speeds up the process of collection, identification, evaluation, and transfer (Fleury and Alvarenga 1999). Thus, it minimizes the risk for people involved in the procedure, the stress and risk for the donor for reducing the time and intensity of transrectal manipulation of the uterus, the cost of material and time of exposure of the embryo to unfavourable conditions. Besides, it optimizes the time of the professional, which is crucial because of the volume of activities involved in an ET programme with a large number of donors. The results from embryo recovery rate from the first to the ninth collections of the same donor in the same breeding season, showed in Table 2, demonstrated that, at least in the short term, frequent manipulation associated with the ET programme does not cause damage to the donor mares’ fertility. The results concerning the use of donor’s foal heats (Table 3) were similar to those obtained by Carvalho et al. (2001a); there were no differences in embryo recovery rates between mares mated in the foal heat or in another heat (57.9% vs 50.0%, respectively), although that author obtained a lower recovery rate. Similarly, the results of the present study are higher than the values obtained by Huhtinen et al. (1996), that reported embryo recovery rates of 48 (11 ⁄ 23) and 71% (10 ⁄ 14), for foal heat and for subsequent cycles, respectively, although these values were not significantly different (p > 0.05), possibly because of the small number of observations. Although the number of donor mares inseminated during foal heat was very low in this study, the 78.6% embryo recovery rate suggests that pregnancy rates are quite normal in this mare category. This results, when compared to natural breeding management, are higher than those reported by Almeida et al. (1995), that  2010 Blackwell Verlag GmbH

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evaluated matings in the foal heat and obtained 60% of pregnancy (15 ⁄ 25). Camillo et al. (1997) also reported similar results, with 72.1% pregnancy rate on 176 foal heats. The use of foal heat allows the rapid entry of the donor in the programme, anticipating the attainment of the targets set for each mare in the breeding season. The only inconvenient for the use of foal heat may be the size of the uterus in mares that do not have good uterine involution, which makes uterine flushing and embryo recovery more difficult. However, Carvalho (2000) reported no difference in the duration of collection or in the fluid recovery between mares in foal heat and single mares. Small variations in pregnancy rates (p < 0.05) between seasons were registered in the present study (Table 4), and results close to those reported on Brazilian commercial ET programmes were observed (Fleury et al. 1989; Jacob et al. 2002). However, the variation was smaller and the values of pregnancy rates were higher than those observed by Squires et al. (1999) for non-surgical transfers (50–75%). These rates show that ET can be done commercially, even subjected to natural obstacles observed at the field, maintaining results comparable to those obtained during experimental conditions. Those differences observed relate to the level of practice of the professional, quality of management, and installations in several horse breeding farms. Decreased results in the 2001 ⁄ 2002 season, which apparently goes against the tendency of improving of the professional, can be explained by the difficulties registered in the onset of the programme, such as the fact that the farm has not been modified to sustain the changes necessary for the establishment of an ET commercial programme. Thus, with the improvement of conditions in the farm, there was an increase (p < 0.05) in pregnancy rate during the following season (2002 ⁄ 2003), which remained at the same level in the 2003 ⁄ 2004 season in other farms which had already preconditions for successful development of such a programme. In this study, the pregnancy rates of 7, 8, and 9 day embryos were 84% (42 ⁄ 50), 79.2% (415 ⁄ 567), and 71.3% (132 ⁄ 185), respectively, with no statistical difference between them. Concerning the results of the pregnancy rates and early embryonic loss at days 15, 30, 45, and 60 after ovulation of the donor, respectively (Table 4), Carnevale et al. (2000) verified pregnancy rates of embryos transferred at days 6, 7, 8, and 9, observing on day 12 of post-ovulation of the donor, rates of 58.5%, 72.5%, 71.6%, and 65.2%, respectively, and on day of 50 postovulation, rates of 54.3%, 66.3%, 55.6%, and 55.4%, respectively, resulting in early embryonic loss rates of 7.3%, 8.6%, 22.4%, and 15.1%, respectively. This suggests that transfer of embryos at days 6 and 7 or the recipient’s oestrous cycle result in a minor embryonic loss. Embryonic loss rates reported in this study are consistent to those previously described for fertile mares (5–24%; Ball 1993), showing that ET does not cause damage to the reproductive efficiency in horses. In  2010 Blackwell Verlag GmbH

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addition, post-transfer pregnancy rates in the present study and other studies are comparable to those reported for post-insemination with fresh semen (40– 79%) (Amann and Pickett 1987). Recipient mares play a key role in the success of the ET programme. For that to occur, it is important to have a good life quality. The absence of this condition can often result in failures and losses. By representing the most populated category within the production system, it is usually impossible for recipient mares to have ideal conditions in the areas of central management of the farms. Owing to this fact, it is recommended that the recipients are kept away from areas of more intensive management of the farms, which requires the transport of collected embryos. Equine embryos have been refrigerated and transported (Table 5) successfully in different ways and systems for periods of up to 24 h (McCue et al. 2000). Fleury et al. (2002), comparing various conditions to carry embryos in temperatures ranging from 15 to 18C, obtained a pregnancy rate of 75.3%, with no difference between the various media, indicating the possibility of transporting cooled embryos for more than 18 h. Squires et al. (1992), using transported embryos cooled at 5C, evaluated the pregnancy rates on days 12, 35, and 50, reporting pregnancy rates of 86%, 80%, and 77%, respectively, with an early embryonic loss rate of 6% to 35 days, and 9% to 50 days. Squires et al. (1999), working with cooled embryos transported in the years 1995–1998, reported rates of 68.9% with 12 days, and 63.9% with 50 days after ovulation of the donor, resulting in early embryonic loss of 5%. Carney et al. (1991) reported no difference in pregnancy rates between cooled transported embryos and embryos collected and transferred immediately. To confirm that, Squires et al. (1999) used the interval from collection to ET between twelve and 30 h, and did not observe any difference between pregnancy rates for stored embryos at 5C for 12 h (63.2%), and 24–30 h (58.9%), and those transferred immediately after collection (67.8%). Although in this study the use of old mares in ET was not discussed, Squires et al. (1999) argued that embryonic development and its transport in oviduct of an old mare may be delayed. Thus, embryonic recovery on days eight and nine of post-ovulation may be more appropriate for older mares. Mares with bad reproductive history produce smaller quantities of embryos, and among the likely causes of reduced embryo recovery for these animals, oviductal and uterine diseases may be suggested, besides early embryonic loss. Carney et al. (1991) reported similar early embryonic loss rate (Table 6) between 12 and 50 days (12–15%) for embryos transferred immediately and embryos transported cooled, demonstrating a requirement of synchrony between donor and recipient, that seems to be the same for both types of transfer. Jacob et al. (2002) adopted the same range of synchrony (+1 to )6) between recipient and donor and reported pregnancy rates of 70.3% (270 ⁄ 384) at 14 days and 64.3% (247 ⁄ 384) at 60 days, with early embryonic loss of 6% 60 days after ovulation of the donor.

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Regarding the pregnancy rate, better results were observed for the intervals of )3 and )2 days of synchrony of ovulation of donor and recipient (p < 0.05), and the lowest rates in the intervals of )6 (p < 0.05), with intermediate values in the intervals of )1 and +1, while 0 and +1 did not differ from the range of )6 (p > 0.05). However, Squires et al. (1999) reported similar pregnancy rates for recipient synchrony with the ovulation of +1 and )3 in relation to the donors. Considering day 0 as a sample taken on day 8 of postovulation, the recipients who had higher pregnancy rates (ranges of )3 and )2) were between 5 and 6 days of ovulation. This result confirms those reported in the literature (Carnevale et al. 2000; Jacob et al. 2002), demonstrating the flexibility that ET has suffered in recent years. This result has great importance, as it enables the current practice that separates donors from recipient mares in the programme, which allows better care with the recipients, which is the most important factor within an ET programme.

Conclusion Based on the results obtained in embryo transfer programmes of this study, it is possible to conclude that the first postpartum cycle of donor mares can be used without prejudice to the embryo recovery and to the pregnancy rates. Also, the embryos can be collected with ages between 7 and 9 days, permitting a better adjust in recipients timing. Additionally, embryos larger than 3 mm can be transferred using an adapted device without prejudice to the pregnancy rates. For the transfer to recipient mares, the optimum synchrony with the donor was from )1 to )5 days, relative to the day of ovulation of the donor, and the embryos can be packet and transported at room temperature for short distances without losses in pregnancy rates. Therefore, this flexibilization in the technique provides benefits for commercial programmes of embryo transfer in mares, enabling optimization in the use of recipient mares, cost reduction, and improvements in the management of horse breeding farms. Conflict of interest None of the authors have any conflict of interest to declare.

Author contributions Lopes, Siqueira, and Rocha designed the study. Pinho wrote the article. Guimara˜es is the adviser. Carvalho and Torres are co-advisers.

References Almeida FQ, Fonseca FA, Espechit CJB, 1995: Efeitos da prostaglandina F-2 alfa e da progesterona na eficieˆncia reprodutiva de e´guas mestic¸as no perı´ odo po´s parto. Rev Bras Zootec 24, 652–659. Alvarenga MA, Landim-Alvarenga FC, Meira C, 1993: Modifications in the technique used to recover equine embryos. Equine Vet J Suppl 15, 111–112.

Amann RP, Pickett BW, 1987: Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. J Equine Vet Sci 7, 145–173. Ball BA, 1993: Embrionic death in mares. In: McKinnon AO, Voss JL (eds), Equine Reproduction. Lea & Febiger, Philadelphia, pp. 517–531. Camillo F, Marmorini P, Romagnoli S, Vannozzi I, Bagliacca M, 1997: Fertility at the first post partum estrous compared with fertility at the following estrous cycles in foaling mares and with fertility in nonfoaling mares. J Equine Vet Sci 17, 203–210. Carmo MT, Trinque CLN, Lima MM, Medeiros ASL, Alvarenga MA, 2003: Estudos da incideˆncia de mu´ltiplas ovulac¸o˜es em e´guas da rac¸a brasileiro de hipismo e suas implicac¸o˜es em um programa de transfereˆncia de embrio˜es. Rev Bras Repr Anim 26, 252–254. Carnevale EM, Ginther OJ, 1992: Relationships of age to uterine function and reproductive efficiency in mares. Theriogenology 37, 1101–1115. Carnevale EM, Ramirez EL, Squires EL, Alvarenga MA, Vanderwall DK, McCue PM, 2000: Factors affecting pregnancy rates and early embryonic death after equine embryo transfer. Theriogenology 54, 965–979. Carney NJ, Squires EL, Cook VM, Seidel Jr GE, Jasko DJ, 1991: Comparison of pregnancy rates from transfer of fresh vs cooled transported equine embryos. Theriogenology 36, 23–32. Carvalho GR, 2000: Estudo de alguns aspectos da transfereˆncia de embrio˜es em equ¨inos. Universidade Federal de Vic¸osa, Vic¸osa – MG. 103 p. Tese (Doutorado em Zootecnia). Carvalho GR, Fonseca FA, Silva Filho JM, Ruas JRM, Borges AM, 2001a: Avaliac¸a˜o da utilizac¸a˜o do cio do potro na coleta de embrio˜es. Rev Bras Zootec 30, 1445–1450. Carvalho GR, Silva Filho JM, Fonseca FA, Ruas JRM, Borges AM, 2001b: Influeˆncia da te´cnica de coleta sobre a taxa de recuperac¸a˜o de embrio˜es equ¨inos. Arq Bras Med Vet Zootec 53, 231–239. Fleury JJ, Alvarenga MA, 1999: Effects of collection day on embryo recovery and pregnancy rates in a nonsurgical equine embryo transfer program. Theriogenology 51, 261. Fleury JJ, Costa Neto JBF, Alvarenga MA, 1989: Results from an embryo transfer programm with Mangalarga mares in Brazil. Equine Vet J Suppl 8, 73–74. Fleury JJ, Fleury PDC, Landin-Alvarenga FC, 2002: Effect of embryo diameter and storage period on pregnancy rates obtained with equine embryos stored in Ham’s F-10 with Hepes Buffer at a temperatute of 15–18C – preliminary results. Theriogenology 58, 749–750. Gomes GM, Jacob JCF, Domingues IB, 2004: Utilization of mares after first post partum ovulation as embryo recipients. In: International Symposium on Equine Embryo Transfer, 6 Rio de Janeiro, Brasil. Proceedings… pp. 12. Huhtinen M, Reilas T, Katila T, 1996: Recovery rates and quality of embryos from mares inseminated at the first postpartum oestrus. Acta Vet Scand 37, 343–350. Jacob JCF, Domingues IB, Gastal EL, Gastal MO, Silva AG, Mello CM, Gasparetto F, 2002: The impact of degree of synchrony between donors and recipients in a commercial equine embryo transfer program. Theriogenology 57, 545. Lopes EP, 2002a: Desmistificando a transfereˆncia de embrio˜es I. Top 2000 Mangalarga Marchador 1, 6. Lopes EP, 2002b: Desmistificando a transfereˆncia de embrio˜es II. Top 2000 Mangalarga Marchador 1, 22. Malschitzky E, Schilela A, Mattos ALG, Garbade P, Gregory RM, Mattos RC, 2002: Effect of intra-uterine fluid accumulation during and after foal-heat and of different management techniques on the postpartum fertility of thoroughbred mares. Theriogenology 58, 495–498.  2010 Blackwell Verlag GmbH

Reproduction in Mangalarga Marchador Mares McCue PM, Scoggin CF, Meira C, Squires EL, 2000: Pregnancy rates for equine embryos cooled for 24 h in Ham’s F-10 versus EmCareTM embryo holding solution. In: Proceedings of the Annual Conference of Society of Theriogenology, pp. 147. Merkt H, Klug E, Jochle W, 2000: Reproduction management in the german thoroughbred breeding industry. J Equine Vet Sci 20, 195–201. Pessoˆa MA, Rocha Filho AN, Carmo MT, 2004: Comparison between Short and Long Acting for Non-Cycling Embryo Recipient Mares. In: International Symposium on Equine Embryo Transfer, 6 Rio de Janeiro, Brasil. Proceedings… pp. 17. Reilas T, Huhtinen M, Oksanen M, Katila T, 2000: Relationship between embryo recovery rate and uterine lavage fluid composition in postpartum mares. Reprod Nutr Dev 40, 383–391. SAEG, 2002: Sistemas de Ana´lises Estatı´ sticas e Gene´ticas. UFV, Vic¸osa-MG. Silva LA, 2003: Te´cnica ultra-sonogra´fica de injec¸a˜o intrauterina para transfereˆncia de embrio˜es em equ¨inos. Universidade Federal de Vic¸osa, Vic¸osa – MG. 123p. Dissertac¸a˜o (Mestrado em Zootecnia).

 2010 Blackwell Verlag GmbH

267 Squires EL, 1993: Embryo transfer. In: McKinnon AO, Voss JL (eds), Equine Reproduction. Philadelphia, Lea & Febiger, pp. 357–367. Squires EL, McKinnon AO, Carnevale EM, Morris R, Nett TM, 1987: Reproductive characteristics of spontaneous single and double ovulating mares and superovulated mares. J Reprod Fertil Suppl 35, 399–403. Squires EL, Cook VM, Jasko DJ, Tarr SF, 1992: Pregnancy rates after collection and shipment of equine embryos (1988–1991). Am Assoc Equine Pract 38, 609–618. Squires EL, McCue PM, Vanderwall D, 1999: The current status of equine embryo transfer. Theriogenology 51, 91–104. Squires EL, Carnevale EM, McCue PM, Bruemmer JE, 2003: Embryo technologies in the horse. Theriogenology 59, 151– 170. Submitted: 21 Aug 2009; Accepted: 4 May 2010 Author’s address (for correspondence): Roge´rio Oliveira Pinho, Universidade Federal de Vic¸osa ⁄ UFV–PH Rolfs Avenue, 36570–000 Vic¸osa City, MG, Brasil. E-mail: [email protected]