Induction of double ovulation in mares using deslorelin acetate

Animal Reproduction Science 136 (2012) 69–73

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Induction of double ovulation in mares using deslorelin acetate J.F. Nagao a , J.R. Neves Neto b , F.O. Papa a , M.A. Alvarenga a , C.P. Freitas-Dell’Aqua a , J.A. Dell’Aqua Junior a,∗ a College of Veterinary Medicine e Animal Science – UNESP University of Sao Paulo State – Department of Animal Reproduction and Veterinary Radiology – Botucatu-SP-Brasil, Rubião Junior District sn, ZP 18600-000, Brazil b Institute of Animal Reproduction, São José dos Pinhais, SP, Brazil

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Article history: Received 26 August 2011 Received in revised form 2 October 2012 Accepted 5 October 2012 Available online 1 November 2012 Keywords: Mare Deslorelin acetate Double ovulation Embryo transfer

a b s t r a c t This study aimed to determine whether deslorelin acetate could induce double ovulation in mares. In Experiment 1, eight mares were treated with prostaglandin on Day 8 (D8) after ovulation, then treated with saline or with 100 ␮g of a controlled-release formulation of deslorelin acetate vehicle intramuscularly (IM) every 12 h from D8 after ovulation until at least two follicles reached 33 mm. At this time, ovulation was induced with 2500 IU of hCG. Artificial insemination was performed 24 h after induction, and embryos were collected on the eighth day after ovulation was first detected. In Experiment 2, 112 estrous cycles in 56 mares were studied. In this experiment, the deslorelin acetate protocol was initiated only in mares that achieved a follicle with a diameter of at least 25 mm and at least one second follicle with a diameter ≥ 20 mm was detected, at which time 100 ␮g deslorelin acetate or saline was administered IM every 12 h. The other procedures were similar to those described in Experiment 1. The variables studied were analyzed using Student’s t-test and Fisher’s exact test. In Experiment 1, only two mares in deslorelin group having second follicles of 20–25 mm on responded with double ovulation. In the second experiment, 82% of treated mares responded with double ovulation, and the embryo recovery per estrous cycle was 1.12 and 0.57 in the group treated with deslorelin acetate and the control group, respectively (P < 0.05). Deslorelin acetate is effective in inducing double ovulation in mares using the protocol proposed. On average, it allows for the recovery of one embryo by uterine flushing. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Currently, most embryo collections in horses are from unique and spontaneous ovulations, resulting in an embryo recovery rate of around 60% (Alvarenga et al., 2008). One method to increase the efficiency of horse embryo transfer is to increase the number of embryos recovered per donor

∗ Corresponding author at: FMVZ – UNESP Botucatu – Department of Animal Reproduction and Veterinary Radiology – Botucatu-SP-Brasil, Rubião Junior District sn, ZP 18600-000, Brazil. Tel.: +55 14 38116249. E-mail address: [email protected] (J.A. Dell’Aqua Junior). 0378-4320/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.anireprosci.2012.10.015

through the induction of multiple ovulations (Squires et al., 2003). A mare ovary has unique features compared to other species, including its larger size and the inverted position of the layers of the cortex and medulla. Although superovulation can be performed in horses using FSH-e the average number of ovulations achieved is 3.4–5.2 (Niswender et al., 2003; Logan et al., 2007; Raz et al., 2009). Alvarenga et al. (2001) used EPE and demonstrated an improvement in the percentage of ovulation, with an average of 4–7 ovulations per mare, but with poor embryo recovery rates. According to this anatomical organization, the follicles can only ovulate through the ovulation fossa (Stabenfeldt et al., 1975; Hirano et al., 2009).

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Possibly as a result of the single site of ovulation, Carmo et al. (2006) found that the reduction in the rate of embryo recovery from superovulated mares may be due to interference in the capture and transport of oocytes through the oviduct by the formation of a large blood clot in the ovulation fossa. This was particularly observed in mares with multiple ovulations in the same ovary. A significant decrease in the number of oocytes/ovulation was also observed when there were ovulations from more than three follicles. Therefore, a protocol to induce ovulation of only two follicles per ovary may provide the most efficient method for multiple ovulations in the mare. In a previous study, we found that two applications of deslorelin acetate can promote double ovulation in mares. The purpose of this study was, therefore, to assess the effect of deslorelin acetate on the induction of double ovulations in mares. 2. Materials and methods 2.1. Experiment 1 We initially used 16 estrous cycles of eight purebred Arabian mares between 3 and 22 years of age, weighing between 350 and 500 kg. In this experiment, all mares received 5 mg of dinoprost tromethamine (Lutalyse® , Pfizer, New York, NY, USA) on Day 8 (D8) of the estrous cycle (D0 = ovulation) and were randomly divided into the control group (n = 4) and treated group (n = 4). In the subsequent estrous cycle, mares were placed in the opposite group. Thus, all mares were treated in one estrous cycle and not treated in the other cycle as an internal control. For the treated group, immediately after the induction of luteolysis on D8, treatment was initiated with 100 ␮g of deslorelin acetate (Bachem, Los Angeles, USA), which was diluted in a polyethyleneglicol vehicle for slow controlled release and given every 12 h via intramuscular (IM) injection, this dose was determined in a previous experiment where it was found this dose promoted growth of two pre-ovulatory follicles per estrous cycle in the same mares. The control group received 1-mL IM injections of a saline solution. The treatment lasted until at least two follicles reached a minimum of 33 mm or until there was no follicular growth for 3 consecutive days. At this point, ovulation was induced with 2500 IU of human chorionic gonadotropin (hCG, Vetecor, Calier Laboratory, São Paulo, Brazil) intravenously only in mares having pre-ovulatory follicles greater than 33 mm as detected by transrectal ultrasonic assessment that was performed every 6 h. Artificial insemination was performed 24 h after the induction of ovulation with 1 × 109 sperm from a stallion known to be fertile, and an uterine flushing was performed 8 days after the ovulation. 2.2. Experiment 2 This experiment used 112 estrous cycles of 56 mares of different breeds between 3 and 22 years of age and weighing 350–500 kg. The animals were divided into control and treated groups, as described in Experiment 1.

After luteolysis induction on D8, the mares were monitored daily using transrectal ultrasonography. When the largest follicle in the ovaries was no larger than 25 mm diameter and the second follicle was at least 20 mm, administration of deslorelin acetate (100 ␮g) or 1 mL saline IM was started at 12 h intervals. Each was administered until at least two follicles reached ≥ 33 mm in diameter or until the largest follicle achieved 38 mm. In the control group follicular growth was stimulated in a single follicle when a size of between 35 and 38 mm. Treatment to induce ovulations was subsequently administered and transrectal ultrasonography was performed every 6 h as described for Experiment 1. Twenty-four hours after administration of hCG, artificial insemination was performed with 1 × 109 sperm from one of five stallions known to be fertile, and embryo collection was performed 8 days after the first ovulation was detected.

2.3. Statistical analysis Statistical analysis was performed using the program SAS, version 9.1. The variables were analyzed using Student’s t-test or Fisher’s exact test. All statistics were considered significant when a P < 0.05 was detected.

3. Results 3.1. Results from Experiment 1 The diameter of the largest follicle at the beginning of the treatment (D8) ranged from <10 to 27 mm in the control group and from <15 to 24 mm in treated group. The diameter of the second largest follicle ranged from <10 to 23 mm in control group and <10–21 mm for treated group. Two mares in the treated group had ovulations from two follicles. These mares both had two follicles with diameters between 20 and 24 mm at the start of the treatment (Table 1). The number of days from the start of treatment until the time of ovulation induction varied from 2 to 11 days in the control group and from 3 to 6 days in the treated mares. Three mares in the treated group showed no follicular growth for 3 consecutive days, and treatment was stopped in those cases (Table 1). These mares had follicular growth until both largest follicles in the ovaries were about 30 mm in diameter, about 4 days after the beginning of the treatment and follicular growth was not observed during the next 3 days. The size of the largest follicle at the time of ovulation induction varied similarly for the both control and treated groups from 33 to 37 mm in diameter (Table 1). There was one ovulation per mare in the control group, whereas two mares in the treated group had double ovulation, three mares had simple ovulation, and three mares not showed follicular growth for three consecutive days and did not have ovulations, these mares were not induced the ovulation (Table 1). Five embryos were recovered from the control group and six from the treated group. Two embryos were

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Table 1 Size (in mm) of the two largest follicles on D8 of the estrous cycle, number of days from treatment until ovulation induction, follicle size (in mm) at the time of induction, number of ovulations per mare and number of embryos recovered. Mares

Control

Treated

SZ (mm) of the 2 largest fols (D8)

Days before induction

SZ (mm) fol(s) at induction

Number of ovulations

Number of embryos recovered

17–20 18–18 <10–<10 <10–<10 23–26 16–18 15–16 20–27 10–18 16–17 15–16 18–16 21–24 20–24 15–18 10–15

5 9 8 11 5 7 7 2 NR 6 NR 6 3 3 5 NR

36 35 36 33 36 35 37 36 – 36 – 33 34–37 33–36 36 –

1 1 1 1 1 1 1 1 0 1 0 1 2 2 1 0

0 1 1 1 0 1 1 0 0 1 0 1 2 2 0 0

D8, Day 8 of the estrous cycle; SZ, size (mm); fol(s), follicle(s); NR, mares that did not show follicular growth for 3 consecutive days.

recovered from the same uterine lavage for the two mares that had two ovulations (Table 1). 3.2. Results from Experiment 2 The average number of days of deslorelin acetate administration until ovulation induction was 3.5 days. The mares in the control group took an average of 6.7 days to ovulation induction (Table 2). The size of the follicles at the time of ovulation induction was similar between the two groups. The interval between induction and the first ovulation was less in the treated group than in the control group (Table 2). There were only four mares that did not have two follicles between 20 and 25 mm during the estrous cycle, and data from these cycles were not utilized for analyses, but the mares were used for study in the subsequent estrous cycle. The interval between ovulations in the treated group was 13.5 h on average. Ovulations were considered asynchronous when they were observed with more than 6 h of difference between examinations (Table 2). The number of mares that had ovulations from two follicles per estrous cycle was greater in the treated group (46 mares, 82%, compared to 0 in control group) (Table 2). None of the mares in the control group had two follicles over 33 mm during the estrous cycle. Ten mares in the treatment group had a single ovulation per estrous cycle and there was no evident growth of the second follicle. The number of ovulations, average number of ovulations per estrous cycle, number of embryos recovered and the number of recovered embryos per estrous cycle were greater in the treated group (102 ovulations; 1.82 ovulations per cycle, 63 embryos; 1.12 embryos recovered per cycle) than in the control group (56 ovulations; 1.0 ovulations per cycle; 32 embryos; 0.57 embryos recovered per cycle) (Table 2). The number and percentage of embryos recovered per ovulation were not different between groups (Table 2).

The proportion of double ovulations occurring from the same ovary (unilateral) was greater than those with bilateral ovulations. However, the rate of embryo recovery did not differ and was independent of the laterality of ovulation (Table 3). Table 2 Absolute or average data and standard deviations for the variables measured between the control group and the group treated with deslorelin acetate to obtain multiple ovulations. Variables

Control

Number of cycles utilized Time to treatment after application of PGF2␣ (days) Diameter of the largest follicle at the initiation of treatment (mm) Diameter of the second largest follicle at the initiation of treatment (mm) Days until ovulation inductiona Days of treatment to ovulation Size(s) of largest follicle(s) at induction Size of the 2nd largest follicle at induction Interval between induction and the first ovulation (hours) Interval between ovulations (hours) of mares ovulating asynchronously Number (%) of mares ovulating synchronously Number (%) of mares that ovulated 2 follicles per cycle Number of ovulations Average number of ovulations per cycle Number of embryos recovered Percentage of embryos recovered per cycle Percentage of embryos recovered per ovulation

56 2.3 (±1.1)

56 2.3 (±1.0)

24.1 (±0.9)

23.7 (±1.0)

22.1 (±1.6)

21.0 (±1.0)

6.75 (±2.8)* 8.5 (±2.9)* 36.3 (±1.7)

3.5 (±0.9) 4.9 (±1.1) 36.7 (±1.9)

– 42 (±5.1)*

Treated

33.8 (±0.6) 35 (±6.1)

0.0 (±0.0)

13.5 (±11.9)

0 (0.0)

37 (80.5)

0* ((0.0)

46 (82)

56* 1.0 (±0.0)*

102 1.82 (±0.5)

32* 57.0*

63 112.5

57

61

a Until two follicles over 33 mm or until the largest follicle was 38 mm and 35–38 mm in control group. * P < 0.05.

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Table 3 Percentage of laterality and the embryo recovery rate from mares that presented with multiple ovulations. Unilateral Laterality (%) Embryo recovery per ovulated follicle (%) *

*

75.0 60.5

Bilateral 25.0 62.5

P < 0.05.

4. Discussion Three animals from Experiment 1 had no follicular growth for three consecutive days. The treatment with deslorelin acetate in these animals lasted 7 days, because after the fourth day of treatment, the follicles stopped growing and maintained the same diameter for three more days. These follicles then regressed, and ovulation did not occur. In several species, continuous administration of large doses of a gonadotropin-releasing hormone (GnRH) agonist induces an initial hyper-secretion of gonadotropins and a subsequent desensitization of the pituitary gland, promoting a reduction in the release of gonadotropin, an event termed, “downregulation” (Irvine and Alexander, 1993). Because three of these animals developed a similar pattern, compatible with downregulation, the treatment protocol with deslorelin acetate was modified to minimize the number of applications per treatment by starting deslorelin treatment when the follicles were larger. The substance tested in the present study produces consistent results, as seen in Experiment 2, compared to substances based on gonadotropins, such as equine pituitary extract (EPE) (Alvarenga et al., 2001; Scoogin et al., 2002). Moreover, deslorelin acetate is a synthetic drug, so it has little to no lot-to-lot variation. Studies (Ginther and Bergfeldt, 1990; Harrison et al., 1990; Fitzgerald et al., 1993; Hyland, 1993; Newcombe et al., 2002; Raz et al., 2009) where GnRH was used in anestrous mares to induce estrous cyclicity provide evidence that there are multiple ovulations in some animals with such treatment regimens. In present study, when applications of deslorelin acetate were started when there were at least two follicles with diameters ranging between 20 and 24 mm, double ovulation occurred in the majority of mares receiving this treatment (82%). In these animals, it is clear that treatments for a shorter period of time (3 days) minimize the likelihood of triggering the downregulation phenomenon and allows for the occurrence of multiple ovulations during the estrous cycle. A similar strategy of reducing the duration of the protocols for inducing multiple ovulations has been reported by Gimenes et al. (2012), who observed that the duration of drug administration decreases when the treatment is initiated when follicles had a diameter of 20–23 mm. The interval between induction of follicular growth and the first ovulation in the mares in the treated group was less compared to the control group. This likely occurred because after the hCG application occurred in the treated group there were two follicles which likely resulted in greater estrogen concentrations, thus inducing a positive feedback

for the preovulatory LH surge. The amount of LH necessary to promote ovulation was, therefore, likely to have occurred earlier than that for the control group. The protocol used in Experiment 2 promoted a lesser ovarian stimulation compared to studies using EPE, where the number of pre-ovulatory follicles varied from 4.7 to 7.1 (Alvarenga et al., 2001; Scoogin et al., 2002). However, the embryo recovery per ovulation was similar per follicle from which ovulation occurred as compared to those obtained in the control group; in contrast with studies that have shown greater numbers of ovulations from follicles, and a lesser embryo recovery per follicle from where ovulations occurred (Alvarenga et al., 2001; Carmo et al., 2006; Scoogin et al., 2002). The presence of multiple pre-ovulatory follicles in the ovary was the explanation offered for the poor rate of embryo recovery found by Carmo et al. (2006), who observed that a large number of ovulations may interfere with the uptake and transport of oocytes via the oviduct after ovulation occurs, due to the formation of a large blood clot in the ovulation fossa. These authors concluded that when more ovulations occur, the recovery rate of oocytes from the oviduct is less. This fact is mainly related to the specific features of the mare’s ovary, in which the positions of the layers of the cortex and medulla are inverted; thus, the follicles can only ovulate through the ovulation fossa (Hirano et al., 2009). Carmo et al. (2006) suggest a maximum of two ovulations per ovary so that the embryo recovery per follicle ovulated is not affected; a similar result was observed in the present experiment. The percentage of mares that had unilateral ovulations was greater than the mares that had bilateral ovulations, corroborating the findings of Duarte (2003). However, the results from the present study differ from those of Riera et al. (2006) where there were no differences in the percentages of unilateral and bilateral ovulations in mares with spontaneous multiple follicle development, after ovulation induction. The rate of embryo recovery in relation to laterality did not differ, which is inconsistent with the findings of Riera et al. (2006) where a greater embryo recovery in mares with bilateral multiple ovulations occurred. According to Alvarenga et al. (2008), the typical rate of embryo recovery per estrous cycle is 60%. Thus, with a 60% rate of pregnancy for embryo transfer, it takes three estrous cycles per embryo donor to achieve a pregnancy. The present study presents a protocol that allows for the recovery of one embryo per estrous cycle, which reduces the cost of embryo transfer programs and increases the probability of achieving a pregnancy.

5. Conclusion Deslorelin acetate is effective in inducing double ovulation in mares when the protocol is initiated when there are at least two follicles between 20 and 25 mm in the ovaries. On average, this protocol doubled the efficiency of embryo transfer by doubling the number of embryos recovered per estrous cycle.

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