- Email: [email protected]
Superovulation, embryo recovery, and pregnancy rates from seasonally anovulatory donor mares treated with recombinant equine FSH (reFSH)
Journal Pre-proof Superovulation, embryo recovery, and pregnancy rates from seasonally anovulatory donor mares treated with recombinant equine FSH (reFSH)
Janet F. Roser, Maria V. Etcharren, Marcelo H. Miragaya, Adrian Mutto, Mark Colgin, Luis Losinno, Pablo J. Ross PII:
S0093-691X(19)30484-4
DOI:
https://doi.org/10.1016/j.theriogenology.2019.10.030
Reference:
THE 15225
To appear in:
Theriogenology
Received Date:
06 August 2019
Accepted Date:
28 October 2019
Please cite this article as: Janet F. Roser, Maria V. Etcharren, Marcelo H. Miragaya, Adrian Mutto, Mark Colgin, Luis Losinno, Pablo J. Ross, Superovulation, embryo recovery, and pregnancy rates from seasonally anovulatory donor mares treated with recombinant equine FSH (reFSH), Theriogenology (2019), https://doi.org/10.1016/j.theriogenology.2019.10.030
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Journal Pre-proof
Superovulation, embryo recovery, and pregnancy rates from seasonally anovulatory donor mares treated with recombinant equine FSH (reFSH) Janet F. Roser, PhD MSa, Maria V. Etcharren, DVMb, Marcelo H. Miragaya, DVM MSc PhDc, Adrian Mutto BS MSc PhDd, Mark Colgin, PhDe#, Luis Losinno, DVM PhDb, Pablo J. Ross, DVM PhD*a Authors’ addresses a. Department of Animal Science, University of California, Davis, CA, USA b. Facultad de Agronomía y Veterinaria, Universidad Nacional de Rio Cuarto, Argentina c. Facultad de Ciencias Veterinarias, INITRA, Universidad de Buenos Aires, Argentina, d. Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martin, Argentina, e. Aspen Bio Pharma Inc., Castle Rock, CO, USA
Key Words: reFSH, anestrus, superovulation, embryo transfer, pregnancy *Corresponding author: Pablo J. Ross, Department of Animal Science, One Shields Ave, University of California, Davis, CA 95616. Fax: (530) 752-0175. email: [email protected] #Current
address: Ceva Animal Health, Lenexa, KS, USA
Journal Pre-proof
Abstract The effectiveness of different treatments with recombinant equine FSH to stimulate follicular growth, multiple ovulations and embryo production in seasonally anovulatory mares was evaluated. During mid-winter season (July-August in Argentina, South America) forty light breed donor mares, presenting follicles <10 mm in diameter and no CL at ultrasound examination (deep-anestrus), were randomly assigned (n=10/group) to one of the following treatments: Group 1: twice daily intramuscular (IM) injections of 0.65 mg reFSH (AspenBio Pharma, CO), Group 2: once daily IM injection of 1.3 mg reFSH, Group 3: twice daily IM injection of 0.32 mg reFSH, and Group 4: once daily IM injection of saline (control). Treatment was administered until a follicle of 35 mm was observed or for a total period of 10 days. When the largest follicle reached ≥ 35 mm in diameter, treatment was discontinued and 2500 IU hCG was injected intravenously (IV) 36 h later. Mares receiving hCG were inseminated with fresh semen every 48 hours until ovulation(s) were detected or one dose of frozen semen (250 x106 motile sperm) after the first ovulation was detected. Eight days after first ovulation, transcervical embryo recovery was performed. Recovered embryos were non-surgically transferred to anovulatory estrogen/progesterone treated recipients and pregnancy diagnosed by ultrasonography 7, 14 and 21 days later. All mares receiving reFSH, but none receiving saline control, responded to the treatment with follicular growth. On average, 6.5 days of reFSH treatment were required for mares to develop follicles of ovulatory size (>35 mm). Ovulations were detected in 80% of mares in Groups 1 and 2, 50% of mares in Group 3 and in none of Group 4 (Control). Among ovulating mares, no differences in number of ovulations, number of embryos recovered, or pregnancy rates were observed among
Journal Pre-proof
reFSH treatments. Of treated mares, 6, 7, and 5 produced embryos in Groups 1, 2, and 3, respectively. The average embryo recovery rate per ovulated mare was 88%. The average embryo recovery rate per ovulation was 43%. Overall, a 59% pregnancy rate was achieved. These results indicate that treatment with reFSH during deep anestrus results in follicular development, ovulation of fertile oocytes, and production of embryos that established viable pregnancies after transfer. Also, a single daily administration of reFSH was as effective as two daily administrations, which allows for a simplified administration regimen. 1. Introduction Embryo transfer (ET) is a widely utilized assisted reproductive technique for mares to obtain the following: a. foals from show mares in competition, b. multiple foals from the same mare in a single year, c. foals from mares with non-reproductive health or musculoskeletal problems and d. foals from mares with reproductive problems. The success rate of ET in a commercial operation is based on the percent of embryo recovery rate per ovulation (50-80%) multiplied by the percent of pregnancy rate per transferred embryo (50-80%). Thus, for a given cycle there is a 25-64% chance of obtaining a pregnant recipient [1–5]. Factors that affect embryo recovery include age of the donor mare, quality of the semen, number of ovulations and day of recovery [2,6]. Factors that affect pregnancy rate are management of the donor and recipient mares, age and health of the donor and recipient mares, non-surgical vs surgical techniques, quality and age of the embryo, synchrony of the recipient mare, transportation and storage of the embryo, manual versus cervical forceps techniques and expertise of the clinician [2,4]. Additional
Journal Pre-proof
factors that have limited the success of ET are the narrow window of time for breeding (breeding season only) and the ability to superovulate the mare. In the Northern and Southern Hemispheres, the natural breeding season for mares is April to September or October to May, respectively. At the end of the breeding season, mares enter a state of ovarian inactivity or seasonal anestrus. The goal of many performance horse owners is to begin breeding mares in early February in the Northern Hemisphere or early July in the Southern Hemisphere, a time when most mares maintained under ambient lights are not cycling. The practice of hastening the first ovulation of the year is carried out to produce early foals the following year that have an economic advantage and competitive edge because of their size and maturity over others born later in the year. Historically, an artificial lighting program has been used to advance the first ovulation of the year [7,8]. However, it takes 60-70 days to work [9,10], is labor intensive and expensive, and not always effective or utilized. Therefore, drugs have been utilized but have been less than consistently successful [11]. Pharmacological compounds have included native gonadotropin releasing hormone (GnRH), GnRH agonists (buserelin, deslorelin, goserelin), dopamine antagonists (domperidone, sulpiride), progesterone, progestins, prolactin, and prostaglandins [11]. Disadvantages of these drugs that arose in previous studies were: the necessity of multiple injections, implants or pumps for greater than 10 d, inconsistent success rates in different studies using the same drug, the necessity for follicles to be more than 20-25 mm in diameter, lower ovulation rates in deep-anestrous mares and return to anestrus after treatment [11]. Equine pituitary extracts (EPE) and partially purified equine FSH (Bioniche Animal Health, Athens, GA) have been relatively effective in inducing superovulation in anestrous mares
Journal Pre-proof
[12,13] and transitional mares [14–17], respectively. However, embryo transfer utilizing EPE preparations has been marginally successful when carried out in deep-anestrous mares [18]. Treatment with EPE has been suggested to have negative effects on embryo viability [19]. The author is not aware of the use of eFSH treatment in a successful embryo transfer program in the deep-anestrous mare. Neither EPE nor eFSH are commercially available. Advancing the first ovulation of the year and inducing superovulation at the same time in deep-anestrous mares under ambient lights has the potential for increasing embryo recovery and decreasing the cost of embryo transfer. In the past few years, reFSH has been shown to consistently induce superovulation followed by high pregnancy rates in both cycling and deep-anestrous mares [20–22]. Recombinant eFSH was developed on a single chain platform and is devoid of other hormones and contaminants [23,24]. In one study, it was demonstrated that deep-anestrous mares that did not get pregnant or lost their pregnancy kept cycling or could be retreated successfully [22]. Recombinant eFSH may be commercially available in the future. The objective of this present study was to determine the success rate of treating seasonally deep-anestrous donor mares under natural ambient lights with reFSH to induce superovulation followed by embryo transfer and pregnancy in the Southern Hemisphere.
Journal Pre-proof
2. Materials and Methods 2.1 Animal selection and management 2.2 Donor mares Clinically healthy deep-anestrous mares of light horse breeds between the ages of 5-13 y weighing between 900-1400 lbs were selected for this study. Mares were housed in single large paddocks at two facilities: La Irenita Equine Embryo Transfer Center, Argentina and University of Buenos Aires, Argentina. All the, animals had unlimited access to grass hay that was fed off the ground in a separate area of the paddock and fed once daily with grain. At both facilities, fresh water was provided ad libitum. Animal care was approved by the ethics and animal welfare committee, Rio Cuarto National University, and Buenos Aires University. Mares were determined to be in deep anestrus by transrectal ultrasound (Aloka SSD 500, Aloka Inc, Japan), examination of the reproductive tract and progesterone analysis. Specifically, mares were selected based on their reproductive records to have no more than 20% multiple ovulations per previous seasons, presenting follicles < 10 mm in diameter and no corpus luteum detected when examined by ultrasound once per week for two consecutive weeks prior to the start of the study. The initial examinations were performed during the weeks of July 1-15th. Blood samples were collected once a week from each mare at the time of each preliminary ultrasound examination. A third blood sample was taken from each mare on July 15th, day of initial treatment. Forty mares were randomly assigned to one of the following four treatment groups at each of the test facilities: Group 1: reFSH at 0.65 mg IM twice daily (n = 10), Group 2:
Journal Pre-proof
reFSH at 1.3 mg IM once daily (n = 10), Group 3: reFSH at 0.32 mg IM twice daily (n = 10), or Group 4: control, phosphate buffered saline (PBS) IM once daily (n = 10). 2.3 Recipient mares The recipients at La Irenita Farm and the University of Buenos Aires were clinically healthy anestrous mares between 3-15 years old and weighing between 900-1400 lbs. with no history of reproductive problems or were maiden mares.
All mares were
maintained on mixed grass pasture and fresh water provided ad libitum. To synchronize the recipients with the donors, the mares were intramuscularly treated with estradiol cypionate (ECP Estradiol® - Koning Laboratories, Argentina) during three consecutive days, using decreasing doses of 10 mg, 6 mg and 4 mg beginning the day ovulation was detected in the donor mare. When mares showed evidence of endometrial edema by ultrasonography for at least 3 days, 1500 mg of long-acting progesterone (P4 LA300® - Laboratorios B.E.T., Rio de Janeiro, RJ, Brazil) were intramuscularly injected on the fourth day. 2.4 Study design 2.4.1 Mare treatments Mares were treated with reFSH in PBS given as an intramuscular (IM) injection according to their treatment group. Group 1 received 1.3 ml (0.65 mg) of reFSH twice daily (BID), 8 h apart; Group 2: received 2.6 mL (1.3 mg) of reFSH once daily (SID) in the morning; and group 3 received 0.65 mL (0.32 mg) of reFSH twice daily (BID), 8 h apart. Control mares (Group 4) received 1.3 mL of PBS as an IM injection once daily in the morning. Treatments were administered until either a) the mare developed a follicle ≥ 35
Journal Pre-proof
mm in diameter or b) for a maximum of 10 consecutive days. Ultrasound per rectum examinations were performed daily during the morning treatment period. The diameter and position of the largest follicle and all of those ≥ 30 mm on each ovary were recorded during each ultrasound examination. Mares detected with a follicle ≥ 35 mm in diameter during the treatment period were allowed to coast for 36 h and then subsequently administered 2,500 units of human chorionic gonadotropin (hCG;Ovusyn®- Syntex, Buenos Aires, Argentina ) intravenously (IV) to induce ovulation. Ultrasound examinations were performed and recorded once daily after detection of a follicle ≥ 35 mm in diameter to confirm the day of ovulation. Mares that developed a follicle ≥ 35 mm in diameter and received hCG were bred every 48 h with fresh semen until ovulation(s) were detected or with frozen semen (250 x 106 motile sperm/dose) in the case of three mares, two in group 1 and one in group 3. Eight days after the first ovulation, standard transervical embryo recovery (see below) was performed and the embryos were graded according to McKinnon and Squires [25], harvested and transferred (see below) to naturally synchronized
or anovulatory treated recipients. Pregnancy was diagnosed by
ultrasonography 7, 14 and 21 days later. After embryo recovery, mares received 2 mL sodium cloprostenol (Estrumate®- Intervet, Germany) IM on two consecutive days. 2.5 Embryo transfer 2.5.1 Transcervical embryo recovery Embryo recovery was performed by transcervical uterine flushes at day 8 after ovulation. The flushing media was Ringer lactate sterile solution (average 2 liters per mare). The embryos were evaluated using a stereomicroscope and classified according size and morphology as described previously [25].
Journal Pre-proof
2.5.2 Nonsurgical transfer of embryos Embryos were transferred into a recipient mare between the third and sixth day after the first long-acting progesterone injection (P0). The mares were once more treated with 1500 mg of long-acting progesterone the day of transfer and every 10 days until 100 days of pregnancy. 2.6 Progesterone assay. Concentrations of plasma progesterone were measured by a validated RIA as previously described [19]. Tritiated progesterone (1, 2, 6, 7-3H-progesterone, NET381, specific activity 90-115 Ci/mmol; Perkin Elmer Life Science, Boston, MA) was used as trace. Standards (Q2600; Steraloids, Wilton, NH) ranged from 0.1 to 20 ng/ml. Plasma samples were diluted with PBS-G when necessary to fall within the standard curve. The primary antibody was a sheep anit-progesterone-11alpha-hemisuccinate: BSA (1/13,000 dilution; #8939 Stabenfeldt, UCD). Extraction efficiency for standards and plasma were the same (75%), so no adjustments of the plasma values using the extraction efficiency were necessary. The sensitivity of the assay was 0.2 ng/ml and the intra- and inter-assay coefficients of variation were 4.0% (n=6) and 8.3% (n=2), respectively. 2.7 Statistical analysis Mean ± standard error of the mean (SEM) are presented in Table 1. Quantitative variables, including days of treatment, follicle size, number of ovulations and number of embryos were analyzed using unpaired Student's t‐test. Proportions, including ovulating mares, embryo recovery rate, and pregnancy rate, were compared using the chi‐squared
Journal Pre-proof
test. Analysis was performed in Excel 2016. A p‐value of <0.05 was considered significant. 3.0 Results All mares were considered to be in deep anestrous based on the presence of follicles <10 mm in diameter, no CL at ultrasound examination and plasma concentrations of progesterone < 1 ng/mL for three consecutive weekly examinations. All mares receiving reFSH, but none receiving saline (Control), responded to the treatment with follicular growth, with all mares reaching ovulatory size (>35 mm), except for two mares in group 3 that only achieved follicles of 20 and 25 mm size during their 10-day treatment period. On average, 6.5 d of reFSH treatment were required for mares to develop follicles of ovulatory size (>35 mm). There was no difference in the number of follicles greater than 35 mm in diameter among the groups. Ovulations were detected in 80% of mares in Groups 1 and 2, 50% of mares in Group 3 and none in Group 4 (Control). Of treated mares, 6, 7, and 5 produced embryos in Groups 1, 2, and 3, respectively. Among ovulating mares, no differences in number of ovulations, number of embryos recovered, or pregnancy rates were observed among reFSH treatments. Combined, for mares producing at least one embryo, the average number of embryos recovered was 2.5 embryos/mare, with 6 mares producing 4 to 5 embryos in a single flush. For the 0.65 mg dose BID, 1.3 mg dose SID and 0.32 dose BID, embryo recovery rates per ovulated mare were 75% (6/8), 88% (7/8), and 100% (5/5), respectively. Embryo recovery rates per ovulation were 37%, 47% and 45%, respectively. Considering all the mares that produced at least one embryo, the embryo recovery rate per ovulation based on number of ovulations per mare were, 59% for up to 4 ovulations (n=7 mares), 75% for 5 to 6
Journal Pre-proof
ovulations (n=6 mares), and 27% for more than 6 ovulations (n=5 mares). Combined, mares with 1 to 6 ovulations had an embryo recovery rate of 69% (n=13 mares). Overall, a 59% pregnancy rate was achieved with embryos collected and transferred during the anestrus season. Results are summarized in Table 1. 4.0 Discussion This is the first study to present successful embryo production, transfer and recipient pregnancy after treatment with reFSH from deep-anestrous donor mares. All deep-anestrous mares administered reFSH under natural winter photoperiod exhibited marked follicular development within 5-7 days after the start of treatment. Ovulation rates were 80% for mares receiving 0.65 mg BID and 1.3 mg SID and 50% for the mares receiving 0.32 mg BID. None of the control mares ovulated. The follicular response and ovulation rates for the higher doses (0.65 mg and 1.3 mg) were similar to that reported in previous studies using EPE [12,18,26] in anestrous mares under natural photoperiod or partially purified eFSH [14,15,27] in transitional mares. Follicular response and ovulation rates were numerically lower in mares receiving a lower reFSH dose (0.32 mg BID), which is similar to what was reported for mares treated during the breeding season with 0.35 mg reFSH BID [20]. Taken together these studies suggest that 0.35 mg BID is insufficient to induce a maximal follicular response. Also, the breeding season study indicated that 0.85 mg reFSH BID resulted in lower embryo recovery per flush compared to 0.65 mg BID, which in combination with the current results indicates that a total daily dose of 1.3 mg of reFSH, administered either as a single 1.3 mg injection or in two 0.65 mg injections, is the optimal level of reFSH for inducing follicular development in mares. It is important to note that giving 1.3 mg SID induced the greatest number of ovulations (5.5) suggesting
Journal Pre-proof
that a single administration of reFSH may be as good, if not better, than twice daily administration. The number of follicles and ovulations per ovulated mare were relatively high, especially considering that control mares showed no follicular activity during the treatment period. On average the number of follicles that reached ≥35 mm in diameter was 5.2 and the number of ovulations was 5.0. Similarly, two previous studies carried out on deep anestrous mares given 0.65 mg of reFSH, the average number of follicles and ovulations were 4.0 and 3.6, respectively [21,22]. Moreover, the ovulations induced in deepanestrous mares resulted in viable embryos, although the number of embryos recovered was considerably lower than the number of ovulations. The average number of embryos recovered per ovulated mare is comparable to the embryo recovery found in mares after treatment with partially purified eFSH during the transition period in two studies by Raz and coworkers (average: 2.2) [17,28]. Studies conducted during the breeding season also observed a similar number of follicles, ovulations and embryos recovered after treatment with reFSH [20,29]. Embryo recovery rates/ovulated mare in this study ranged from 75-100% depending on the dose of reFSH, which is higher than what is generally reported for nonstimulated cycles, ranging from 50% to 70% embryo recovery per cycle [30]. Similarly, treatment with eFSH (Bioniche) resulted in a 90% embryo recovery rate/cycle compared to 42% in mares receiving no treatment [31]. However, embryo recovery rates per ovulation, which in this study ranged from 37-47%, are generally lower in superovulated mares compared to non-stimulated mares. Logan and coworkers (2007) reported an embryo recovery rate per ovulation in cycling mares that was lower after treatment with
Journal Pre-proof
eFSH than in controls [32]. Speculation has it that the ovulation fossa prevents multiple oocytes from escaping the ovary into the oviduct [33]. In this study, multiple mares produced 4-5 embryos in a single flush (n=6 mares), which indicates that mares are capable of ovulating at least two fertile oocytes per ovary. The embryo recovery rates were high (75%) for mares ovulating 5-6 follicles, which requires at least one ovary undergoing a minimum of 3 ovulations. On the other hand, mares with more than 6 ovulations presented greatly decreased embryo recovery rates, suggesting that more than 3 ovulations per ovary may be detrimental to oocyte deposition into the oviduct, oocyte quality, fertilization rates, or embryo development. A study by Carmo and coworkers [34] indicated that the number of oocytes collected surgically from the oviducts of superovulated mares with EPE was higher than from control mares. The number of oocytes collected per ovulation was greater for EPE-treated mares with 1-3 ovulations than for EPE-treated mares with more than three ovulations. The oocyte recovery rate in EPE-treated mares with three or fewer ovulations was the same as in untreated control mares. The authors found that the ovulation fossa in the superovulated mares had large amounts of coagulated blood which was not observed in the control mares, and concluded that excessive hemorrhage in multiple-ovulating ovaries could be involved in the failure of the oocytes to enter the oviduct. Further refining of the protocol may increase embryo recovery rates. It is interesting to note that when LH was added to the treatment, there was an increase in embryo recovery per ovulation. Meyers-Brown reported that recovery after treatment with reFSH/reLH was 83% compared to 66% in cycling mares treated with reFSH alone [29]. The effect of LH on the ovulation fossa is unknown but it may enhance oocyte development and maturation [29]. Some of the variations in recovery
Journal Pre-proof
rates may be a function of whether the ovulations were from one ovary or both. Bilateral ovulations may prove to be a better condition for the escape of oocytes through both ovulations fosses resulting in higher embryo recovery per ovulation. The effect of multiple follicular development and multiple ovulations on fertilization rate, oocyte quality and early embryo development has not been addressed, but given the altered hormonal environment resulting from multiple follicles/corpus lutea it is possible that these events may be affected. Pregnancy rates in recipient mares are also quite variable depending on the study. In a large study carried out by Jacob and coworkers (2012), in which donor mares did not receive any stimulatory agent, the average pregnancy rates for recipient mares (nonsurgical transfer) between days +1 to -3 was 69% [35]. The average pregnancy rate for recipient mares using the Wilsher technique (vaginal speculum and cervical grasping forceps) was 92.3% compared to 70.9% using the conventional technique [4]. Recipient pregnancy rate per non-surgically transferred embryo in mares that received eFSH was 33% compared to 67% in control mares [36]. Pregnancy rates in the present study were 56-75% depending on the dose of reFSH received. Both recovery rates and pregnancy rates were quite acceptable considering the time of year this study was carried out. The ovaries of mares in deep anestrus were inactive with ≤ 10 mm in diameter follicles prior to the start of treatment. To be able to stimulate the ovary in 6-7 days is remarkable. Additionally, 14-16 embryos were transferred from superovulated mares compared to 0 in controls.
Journal Pre-proof
5.0 Conclusion These results indicate that treatment with reFSH during deep anestrus results in follicular development, ovulation of fertile oocytes, and production of embryos that established viable pregnancies after transfer. Also, a single daily administration of reFSH was as effective as two daily administrations, which allows for a simplified administration regimen. Acknowledgements Dr Jorge McDonough† and Sebastian Bereterbide from La Irenita Embriones, Daireux, Argentina. AspenBio Pharma provided the reFSH used in this study. References [1]
Squires EL, Carnevale EM, McCue PM, Bruemmer JE. Embryo technologies in the horse. Theriogenology 2003;59:151–70. doi:10.1016/S0093-691X(02)012682.
[2]
Squires EL, McCue PM, Vanderwall D. The current status of equine embryo transfer. Theriogenology 1999;51:91–104.
[3]
Stout TAE. Equine embryo transfer: review of developing potential. Equine Vet J 2006;38:467–78. doi:10.2746/042516406778400529.
[4]
Cuervo-Arango J, Claes AN, Stout TA. Effect of embryo transfer technique on the likelihood of pregnancy in the mare: a comparison of conventional and Wilsher’s forceps-assisted transfer. Vet Rec 2018;183:323–323. doi:10.1136/vr.104808.
[5]
Panarace M, Pellegrini RO, Basualdo MO, Belé M, Ursino DA, Cisterna R, et al. First field results on the use of stallion sex-sorted semen in a large-scale embryo transfer program. Theriogenology 2014;81:520–5. doi:10.1016/j.theriogenology.2013.10.021.
[6]
Panzani D, Rota A, Marmorini P, Vannozzi I, Camillo F. Retrospective study of factors affecting multiple ovulations, embryo recovery, quality, and diameter in a commercial equine embryo transfer program. Theriogenology 2014;82:807–14. doi:10.1016/j.theriogenology.2014.06.020.
[7]
Burkhardt J. Transition from anoestrus in the mare and the effects of artificial lighting. J Agric Sci 1947;37:64–8. doi:10.1017/S0021859600013083.
[8]
Palmer E, Driancourt MA, Ortavant R. Photoperiodic stimulation of the mare
Journal Pre-proof
during winter anoestrus. J Reprod Fertil Suppl 1982;32:275–82. [9]
Scraba ST, Ginther OJ. Effects of lighting programs on onset of the ovulatory season in mares. Theriogenology 1985;24:667–79.
[10] Ginther OJ. Anovulatory season. In: Ginther OJ, editor. Reprod. Biol. Mare. Second edi, Cross Plains, WI: Equiservices; 1992, p. 136–72. [11] McCue PM, Logan NL, Magee C. Management of the transition period: Hormone therapy. Equine Vet Educ 2007;19:215–21. doi:10.2746/095777307X187793. [12] Lapin DR, Ginther OJ. Induction of ovulation and multiple ovulations in seasonally anovulatory and ovulatory mares with an equine pituitary extract. J Anim Sci 1977;44:834–42. doi:10.2527/jas1977.445834x. [13] Coy RE, McCue PM, Bruemmer JE, Squires EL. Follicular response in mares in deep or transitional anestrus to equine pituitary extract. Proc Equine Nutr Physiol Symp 1999;16:65–8. [14] Niswender KD, Mccue PM, Squires EL. Effect of Purified Equine FollicleStimulating Hormone on Follicular Development. J Equine Vet Sci 2004;24:37–9. doi:10.1016/jevs.2003.12.011. [15] Peres KR, Fernandes CB, Alvarenga MA, Landim-Alvarenga FC. Effect of eFSH on Ovarian Cyclicity and Embryo Production of Mares in Spring Transitional Phase. J Equine Vet Sci 2007;27:176–80. doi:10.1016/j.jevs.2007.02.009. [16] Raz T, Hunter B, Carley S, Card C. Reproductive performance of donor mares subsequent to eFSH treatment in early vernal transition: Comparison between the first, second, and mid-season estrous cycles of the breeding season. Anim Reprod Sci 2009;116:107–18. doi:10.1016/j.anireprosci.2008.12.008. [17] Raz T, Amorim MD, Stover BC, Card CE. Ovulation, pregnancy rate and early embryonic development in vernal transitional mares treated with equine- or porcine-FSH. Reprod Domest Anim 2010;45:287–94. doi:10.1111/j.14390531.2008.01296.x. [18] Woods GL, Ginther OJ. Ovarian response, pregnancy rate, and incidence of multiple fetuses in mares treated with an equine pituitary extract. J Reprod Fertil Suppl 1982;32:415–21. [19] Woods GL, Ginther OJ. Intrauterine embryo reduction in the mare. Theriogenology 1983;20:699–706. [20] Meyers-Brown GA, McCue PM, Niswender KD, Squires EL, DeLuca CA, Bidstrup LA, et al. Superovulation in Mares Using Recombinant Equine Follicle Stimulating Hormone: Ovulation Rates, Embryo Retrieval, and Hormone Profiles. J Equine Vet Sci 2010;30:560–8. doi:10.1016/j.jevs.2010.09.007. [21] Meyers-Brown GA, McCue PM, Troedsson MHT, Klein C, Zent W, Ferris RA, et al. Induction of ovulation in seasonally anestrous mares under ambient lights using recombinant equine FSH (reFSH). Theriogenology 2013;80:456–62.
Journal Pre-proof
doi:10.1016/j.theriogenology.2013.04.029. [22] Meyers-Brown GA, Loud MC, Hyland JC, Roser JF. Deep anestrous mares under natural photoperiod treated with recombinant equine FSH (reFSH) and LH (reLH) have fertile ovulations and become pregnant. Theriogenology 2017;98:108–15. doi:10.1016/j.theriogenology.2017.05.001. [23] Jennings MW, Boime I, Daphna-Iken D, Jablonka-Shariff A, Conley AJ, Colgin M, et al. The efficacy of recombinant equine follicle stimulating hormone (reFSH) to promote follicular growth in mares using a follicular suppression model. Anim Reprod Sci 2009;116:291–307. doi:10.1016/j.anireprosci.2009.01.013. [24] Jablonka-Shariff A, Roser JF, Bousfield GR, Wolfe MW, Sibley LE, Colgin M, et al. Expression and bioactivity of a single chain recombinant equine luteinizing hormone (reLH). Theriogenology 2007;67:311–20. doi:10.1016/j.theriogenology.2006.06.013. [25] McKinnon AO, Squires EL. Morphological assessment of the equine embryo. J Am Vet Med Assoc 1988;192:401–6. [26] Douglas RH, Nuti L, Ginther OJ. Induction of ovulation and multiple ovulation in seasonally-anovulatory mares with equine pituitary fractions. Theriogenology 1974;2:133–42. doi:10.1016/0093-691X(74)90063-6. [27] Raz T, Gray A, Hunter B, Card C. Early effects of equine FSH (eFSH) treatment on hormonal and reproductive parameters in mares intended to carry their own pregnancy. Anim Reprod Sci 2009;115:76–87. doi:10.1016/j.anireprosci.2008.11.001. [28] Raz T, Carley S, Card C. Comparison of the effects of eFSH and deslorelin treatment regimes on ovarian stimulation and embryo production of donor mares in early vernal transition. Theriogenology 2009;71:1358–66. doi:10.1016/j.theriogenology.2008.09.048. [29] 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;128:52–9. doi:10.1016/j.anireprosci.2011.09.002. [30] Squires EL. Breakthroughs in Equine Embryo Cryopreservation. Vet Clin North Am - Equine Pract 2016;32:415–24. doi:10.1016/j.cveq.2016.07.009. [31] Niswender KD, Alvarenga MA, McCue PM, Hardy QP, Squires EL. Superovulation in Cycling Mares Using Equine Follicle Stimulating Hormone (eFSH). J Equine Vet Sci 2003;23:497–500. doi:10.1016/j.jevs.2003.10.002. [32] Logan NL, McCue PM, Alonso MA, Squires EL. Evaluation of three equine FSH superovulation protocols in mares. Anim Reprod Sci 2007;102:48–55. doi:10.1016/j.anireprosci.2006.09.027. [33] Allen W. The Development and Application of the Modern Reproductive Technologies to Horse Breeding. Reprod Domest Anim 2005;40:310–29.
Journal Pre-proof
doi:10.1111/j.1439-0531.2005.00602.x. [34] Carmo MT, Losinno L, Aquilar JJ, Araujo GHM. Oocyte transport to the oviduct of superovulated mares. Anim Reprod Sci 2006;94:337–9. doi:10.1016/j.anireprosci.2006.03.080. [35] 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;77:1159–66. doi:10.1016/j.theriogenology.2011.10.022. [36] Raz T, Green G, Carley S, Card C. Folliculogenesis, embryo parameters and post-transfer recipient pregnancy rate following equine follicle-stimulating hormone (eFSH) treatment in cycling donor mares. Aust Vet J 2011;89:138–42. doi:10.1111/j.1751-0813.2011.00691.x.
Journal Pre-proof
Highlights
reFSH stimulates follicular development and ovulation in deep anestrus mares. Once-a-day reFSH administration is sufficient for inducing superovulation. Embryos from reFSH-treated mares establish pregnancy after embryo transfer.
Journal Pre-proof
Table 1. Ovulation and embryo recovery rates of anovulatory mares treated with reFSH Dose of 0.65 mg BID 1.3 mg SID reFSH Days of 5.9 ± 0.6a 7.1 ± 0.8a treatment Ovulating 8/10 8/10 mares Follicles > 35 mm in 5.2 ± 0.9a 6.4 ± 0.8a diameter Ovulations per ovulated 5.1 ± 1.0 5.5 ± 0.8 mare Embryos recovered 1.9 ± 0.5 2.6 ± 0.5 per ovulated mare Embryo recovery 75% 88% rate per (7/8) (6/8) ovulated mare Embryo recovery 37% 47% rate per ovulation Embryos 14 16 transferred Pregnancy rate per 57% 56% transferred embryo Values indicate mean ± SEM a, b: Different letters indicate p < 0.05
0.32 mg BID
Saline control
7.3 ± 0.7a
10 ± 0b
5/10
0/10
4.1 ± 1.0a
0 ± 0b
4.4 ± 0.9
-
2.0 ± 0.4
-
100% (5/5)
-
45%
-
4
-
75%
-
Janet F. Roser, Maria V. Etcharren, Marcelo H. Miragaya, Adrian Mutto, Mark Colgin, Luis Losinno, Pablo J. Ross PII:
S0093-691X(19)30484-4
DOI:
https://doi.org/10.1016/j.theriogenology.2019.10.030
Reference:
THE 15225
To appear in:
Theriogenology
Received Date:
06 August 2019
Accepted Date:
28 October 2019
Please cite this article as: Janet F. Roser, Maria V. Etcharren, Marcelo H. Miragaya, Adrian Mutto, Mark Colgin, Luis Losinno, Pablo J. Ross, Superovulation, embryo recovery, and pregnancy rates from seasonally anovulatory donor mares treated with recombinant equine FSH (reFSH), Theriogenology (2019), https://doi.org/10.1016/j.theriogenology.2019.10.030
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Journal Pre-proof
Superovulation, embryo recovery, and pregnancy rates from seasonally anovulatory donor mares treated with recombinant equine FSH (reFSH) Janet F. Roser, PhD MSa, Maria V. Etcharren, DVMb, Marcelo H. Miragaya, DVM MSc PhDc, Adrian Mutto BS MSc PhDd, Mark Colgin, PhDe#, Luis Losinno, DVM PhDb, Pablo J. Ross, DVM PhD*a Authors’ addresses a. Department of Animal Science, University of California, Davis, CA, USA b. Facultad de Agronomía y Veterinaria, Universidad Nacional de Rio Cuarto, Argentina c. Facultad de Ciencias Veterinarias, INITRA, Universidad de Buenos Aires, Argentina, d. Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martin, Argentina, e. Aspen Bio Pharma Inc., Castle Rock, CO, USA
Key Words: reFSH, anestrus, superovulation, embryo transfer, pregnancy *Corresponding author: Pablo J. Ross, Department of Animal Science, One Shields Ave, University of California, Davis, CA 95616. Fax: (530) 752-0175. email: [email protected] #Current
address: Ceva Animal Health, Lenexa, KS, USA
Journal Pre-proof
Abstract The effectiveness of different treatments with recombinant equine FSH to stimulate follicular growth, multiple ovulations and embryo production in seasonally anovulatory mares was evaluated. During mid-winter season (July-August in Argentina, South America) forty light breed donor mares, presenting follicles <10 mm in diameter and no CL at ultrasound examination (deep-anestrus), were randomly assigned (n=10/group) to one of the following treatments: Group 1: twice daily intramuscular (IM) injections of 0.65 mg reFSH (AspenBio Pharma, CO), Group 2: once daily IM injection of 1.3 mg reFSH, Group 3: twice daily IM injection of 0.32 mg reFSH, and Group 4: once daily IM injection of saline (control). Treatment was administered until a follicle of 35 mm was observed or for a total period of 10 days. When the largest follicle reached ≥ 35 mm in diameter, treatment was discontinued and 2500 IU hCG was injected intravenously (IV) 36 h later. Mares receiving hCG were inseminated with fresh semen every 48 hours until ovulation(s) were detected or one dose of frozen semen (250 x106 motile sperm) after the first ovulation was detected. Eight days after first ovulation, transcervical embryo recovery was performed. Recovered embryos were non-surgically transferred to anovulatory estrogen/progesterone treated recipients and pregnancy diagnosed by ultrasonography 7, 14 and 21 days later. All mares receiving reFSH, but none receiving saline control, responded to the treatment with follicular growth. On average, 6.5 days of reFSH treatment were required for mares to develop follicles of ovulatory size (>35 mm). Ovulations were detected in 80% of mares in Groups 1 and 2, 50% of mares in Group 3 and in none of Group 4 (Control). Among ovulating mares, no differences in number of ovulations, number of embryos recovered, or pregnancy rates were observed among
Journal Pre-proof
reFSH treatments. Of treated mares, 6, 7, and 5 produced embryos in Groups 1, 2, and 3, respectively. The average embryo recovery rate per ovulated mare was 88%. The average embryo recovery rate per ovulation was 43%. Overall, a 59% pregnancy rate was achieved. These results indicate that treatment with reFSH during deep anestrus results in follicular development, ovulation of fertile oocytes, and production of embryos that established viable pregnancies after transfer. Also, a single daily administration of reFSH was as effective as two daily administrations, which allows for a simplified administration regimen. 1. Introduction Embryo transfer (ET) is a widely utilized assisted reproductive technique for mares to obtain the following: a. foals from show mares in competition, b. multiple foals from the same mare in a single year, c. foals from mares with non-reproductive health or musculoskeletal problems and d. foals from mares with reproductive problems. The success rate of ET in a commercial operation is based on the percent of embryo recovery rate per ovulation (50-80%) multiplied by the percent of pregnancy rate per transferred embryo (50-80%). Thus, for a given cycle there is a 25-64% chance of obtaining a pregnant recipient [1–5]. Factors that affect embryo recovery include age of the donor mare, quality of the semen, number of ovulations and day of recovery [2,6]. Factors that affect pregnancy rate are management of the donor and recipient mares, age and health of the donor and recipient mares, non-surgical vs surgical techniques, quality and age of the embryo, synchrony of the recipient mare, transportation and storage of the embryo, manual versus cervical forceps techniques and expertise of the clinician [2,4]. Additional
Journal Pre-proof
factors that have limited the success of ET are the narrow window of time for breeding (breeding season only) and the ability to superovulate the mare. In the Northern and Southern Hemispheres, the natural breeding season for mares is April to September or October to May, respectively. At the end of the breeding season, mares enter a state of ovarian inactivity or seasonal anestrus. The goal of many performance horse owners is to begin breeding mares in early February in the Northern Hemisphere or early July in the Southern Hemisphere, a time when most mares maintained under ambient lights are not cycling. The practice of hastening the first ovulation of the year is carried out to produce early foals the following year that have an economic advantage and competitive edge because of their size and maturity over others born later in the year. Historically, an artificial lighting program has been used to advance the first ovulation of the year [7,8]. However, it takes 60-70 days to work [9,10], is labor intensive and expensive, and not always effective or utilized. Therefore, drugs have been utilized but have been less than consistently successful [11]. Pharmacological compounds have included native gonadotropin releasing hormone (GnRH), GnRH agonists (buserelin, deslorelin, goserelin), dopamine antagonists (domperidone, sulpiride), progesterone, progestins, prolactin, and prostaglandins [11]. Disadvantages of these drugs that arose in previous studies were: the necessity of multiple injections, implants or pumps for greater than 10 d, inconsistent success rates in different studies using the same drug, the necessity for follicles to be more than 20-25 mm in diameter, lower ovulation rates in deep-anestrous mares and return to anestrus after treatment [11]. Equine pituitary extracts (EPE) and partially purified equine FSH (Bioniche Animal Health, Athens, GA) have been relatively effective in inducing superovulation in anestrous mares
Journal Pre-proof
[12,13] and transitional mares [14–17], respectively. However, embryo transfer utilizing EPE preparations has been marginally successful when carried out in deep-anestrous mares [18]. Treatment with EPE has been suggested to have negative effects on embryo viability [19]. The author is not aware of the use of eFSH treatment in a successful embryo transfer program in the deep-anestrous mare. Neither EPE nor eFSH are commercially available. Advancing the first ovulation of the year and inducing superovulation at the same time in deep-anestrous mares under ambient lights has the potential for increasing embryo recovery and decreasing the cost of embryo transfer. In the past few years, reFSH has been shown to consistently induce superovulation followed by high pregnancy rates in both cycling and deep-anestrous mares [20–22]. Recombinant eFSH was developed on a single chain platform and is devoid of other hormones and contaminants [23,24]. In one study, it was demonstrated that deep-anestrous mares that did not get pregnant or lost their pregnancy kept cycling or could be retreated successfully [22]. Recombinant eFSH may be commercially available in the future. The objective of this present study was to determine the success rate of treating seasonally deep-anestrous donor mares under natural ambient lights with reFSH to induce superovulation followed by embryo transfer and pregnancy in the Southern Hemisphere.
Journal Pre-proof
2. Materials and Methods 2.1 Animal selection and management 2.2 Donor mares Clinically healthy deep-anestrous mares of light horse breeds between the ages of 5-13 y weighing between 900-1400 lbs were selected for this study. Mares were housed in single large paddocks at two facilities: La Irenita Equine Embryo Transfer Center, Argentina and University of Buenos Aires, Argentina. All the, animals had unlimited access to grass hay that was fed off the ground in a separate area of the paddock and fed once daily with grain. At both facilities, fresh water was provided ad libitum. Animal care was approved by the ethics and animal welfare committee, Rio Cuarto National University, and Buenos Aires University. Mares were determined to be in deep anestrus by transrectal ultrasound (Aloka SSD 500, Aloka Inc, Japan), examination of the reproductive tract and progesterone analysis. Specifically, mares were selected based on their reproductive records to have no more than 20% multiple ovulations per previous seasons, presenting follicles < 10 mm in diameter and no corpus luteum detected when examined by ultrasound once per week for two consecutive weeks prior to the start of the study. The initial examinations were performed during the weeks of July 1-15th. Blood samples were collected once a week from each mare at the time of each preliminary ultrasound examination. A third blood sample was taken from each mare on July 15th, day of initial treatment. Forty mares were randomly assigned to one of the following four treatment groups at each of the test facilities: Group 1: reFSH at 0.65 mg IM twice daily (n = 10), Group 2:
Journal Pre-proof
reFSH at 1.3 mg IM once daily (n = 10), Group 3: reFSH at 0.32 mg IM twice daily (n = 10), or Group 4: control, phosphate buffered saline (PBS) IM once daily (n = 10). 2.3 Recipient mares The recipients at La Irenita Farm and the University of Buenos Aires were clinically healthy anestrous mares between 3-15 years old and weighing between 900-1400 lbs. with no history of reproductive problems or were maiden mares.
All mares were
maintained on mixed grass pasture and fresh water provided ad libitum. To synchronize the recipients with the donors, the mares were intramuscularly treated with estradiol cypionate (ECP Estradiol® - Koning Laboratories, Argentina) during three consecutive days, using decreasing doses of 10 mg, 6 mg and 4 mg beginning the day ovulation was detected in the donor mare. When mares showed evidence of endometrial edema by ultrasonography for at least 3 days, 1500 mg of long-acting progesterone (P4 LA300® - Laboratorios B.E.T., Rio de Janeiro, RJ, Brazil) were intramuscularly injected on the fourth day. 2.4 Study design 2.4.1 Mare treatments Mares were treated with reFSH in PBS given as an intramuscular (IM) injection according to their treatment group. Group 1 received 1.3 ml (0.65 mg) of reFSH twice daily (BID), 8 h apart; Group 2: received 2.6 mL (1.3 mg) of reFSH once daily (SID) in the morning; and group 3 received 0.65 mL (0.32 mg) of reFSH twice daily (BID), 8 h apart. Control mares (Group 4) received 1.3 mL of PBS as an IM injection once daily in the morning. Treatments were administered until either a) the mare developed a follicle ≥ 35
Journal Pre-proof
mm in diameter or b) for a maximum of 10 consecutive days. Ultrasound per rectum examinations were performed daily during the morning treatment period. The diameter and position of the largest follicle and all of those ≥ 30 mm on each ovary were recorded during each ultrasound examination. Mares detected with a follicle ≥ 35 mm in diameter during the treatment period were allowed to coast for 36 h and then subsequently administered 2,500 units of human chorionic gonadotropin (hCG;Ovusyn®- Syntex, Buenos Aires, Argentina ) intravenously (IV) to induce ovulation. Ultrasound examinations were performed and recorded once daily after detection of a follicle ≥ 35 mm in diameter to confirm the day of ovulation. Mares that developed a follicle ≥ 35 mm in diameter and received hCG were bred every 48 h with fresh semen until ovulation(s) were detected or with frozen semen (250 x 106 motile sperm/dose) in the case of three mares, two in group 1 and one in group 3. Eight days after the first ovulation, standard transervical embryo recovery (see below) was performed and the embryos were graded according to McKinnon and Squires [25], harvested and transferred (see below) to naturally synchronized
or anovulatory treated recipients. Pregnancy was diagnosed by
ultrasonography 7, 14 and 21 days later. After embryo recovery, mares received 2 mL sodium cloprostenol (Estrumate®- Intervet, Germany) IM on two consecutive days. 2.5 Embryo transfer 2.5.1 Transcervical embryo recovery Embryo recovery was performed by transcervical uterine flushes at day 8 after ovulation. The flushing media was Ringer lactate sterile solution (average 2 liters per mare). The embryos were evaluated using a stereomicroscope and classified according size and morphology as described previously [25].
Journal Pre-proof
2.5.2 Nonsurgical transfer of embryos Embryos were transferred into a recipient mare between the third and sixth day after the first long-acting progesterone injection (P0). The mares were once more treated with 1500 mg of long-acting progesterone the day of transfer and every 10 days until 100 days of pregnancy. 2.6 Progesterone assay. Concentrations of plasma progesterone were measured by a validated RIA as previously described [19]. Tritiated progesterone (1, 2, 6, 7-3H-progesterone, NET381, specific activity 90-115 Ci/mmol; Perkin Elmer Life Science, Boston, MA) was used as trace. Standards (Q2600; Steraloids, Wilton, NH) ranged from 0.1 to 20 ng/ml. Plasma samples were diluted with PBS-G when necessary to fall within the standard curve. The primary antibody was a sheep anit-progesterone-11alpha-hemisuccinate: BSA (1/13,000 dilution; #8939 Stabenfeldt, UCD). Extraction efficiency for standards and plasma were the same (75%), so no adjustments of the plasma values using the extraction efficiency were necessary. The sensitivity of the assay was 0.2 ng/ml and the intra- and inter-assay coefficients of variation were 4.0% (n=6) and 8.3% (n=2), respectively. 2.7 Statistical analysis Mean ± standard error of the mean (SEM) are presented in Table 1. Quantitative variables, including days of treatment, follicle size, number of ovulations and number of embryos were analyzed using unpaired Student's t‐test. Proportions, including ovulating mares, embryo recovery rate, and pregnancy rate, were compared using the chi‐squared
Journal Pre-proof
test. Analysis was performed in Excel 2016. A p‐value of <0.05 was considered significant. 3.0 Results All mares were considered to be in deep anestrous based on the presence of follicles <10 mm in diameter, no CL at ultrasound examination and plasma concentrations of progesterone < 1 ng/mL for three consecutive weekly examinations. All mares receiving reFSH, but none receiving saline (Control), responded to the treatment with follicular growth, with all mares reaching ovulatory size (>35 mm), except for two mares in group 3 that only achieved follicles of 20 and 25 mm size during their 10-day treatment period. On average, 6.5 d of reFSH treatment were required for mares to develop follicles of ovulatory size (>35 mm). There was no difference in the number of follicles greater than 35 mm in diameter among the groups. Ovulations were detected in 80% of mares in Groups 1 and 2, 50% of mares in Group 3 and none in Group 4 (Control). Of treated mares, 6, 7, and 5 produced embryos in Groups 1, 2, and 3, respectively. Among ovulating mares, no differences in number of ovulations, number of embryos recovered, or pregnancy rates were observed among reFSH treatments. Combined, for mares producing at least one embryo, the average number of embryos recovered was 2.5 embryos/mare, with 6 mares producing 4 to 5 embryos in a single flush. For the 0.65 mg dose BID, 1.3 mg dose SID and 0.32 dose BID, embryo recovery rates per ovulated mare were 75% (6/8), 88% (7/8), and 100% (5/5), respectively. Embryo recovery rates per ovulation were 37%, 47% and 45%, respectively. Considering all the mares that produced at least one embryo, the embryo recovery rate per ovulation based on number of ovulations per mare were, 59% for up to 4 ovulations (n=7 mares), 75% for 5 to 6
Journal Pre-proof
ovulations (n=6 mares), and 27% for more than 6 ovulations (n=5 mares). Combined, mares with 1 to 6 ovulations had an embryo recovery rate of 69% (n=13 mares). Overall, a 59% pregnancy rate was achieved with embryos collected and transferred during the anestrus season. Results are summarized in Table 1. 4.0 Discussion This is the first study to present successful embryo production, transfer and recipient pregnancy after treatment with reFSH from deep-anestrous donor mares. All deep-anestrous mares administered reFSH under natural winter photoperiod exhibited marked follicular development within 5-7 days after the start of treatment. Ovulation rates were 80% for mares receiving 0.65 mg BID and 1.3 mg SID and 50% for the mares receiving 0.32 mg BID. None of the control mares ovulated. The follicular response and ovulation rates for the higher doses (0.65 mg and 1.3 mg) were similar to that reported in previous studies using EPE [12,18,26] in anestrous mares under natural photoperiod or partially purified eFSH [14,15,27] in transitional mares. Follicular response and ovulation rates were numerically lower in mares receiving a lower reFSH dose (0.32 mg BID), which is similar to what was reported for mares treated during the breeding season with 0.35 mg reFSH BID [20]. Taken together these studies suggest that 0.35 mg BID is insufficient to induce a maximal follicular response. Also, the breeding season study indicated that 0.85 mg reFSH BID resulted in lower embryo recovery per flush compared to 0.65 mg BID, which in combination with the current results indicates that a total daily dose of 1.3 mg of reFSH, administered either as a single 1.3 mg injection or in two 0.65 mg injections, is the optimal level of reFSH for inducing follicular development in mares. It is important to note that giving 1.3 mg SID induced the greatest number of ovulations (5.5) suggesting
Journal Pre-proof
that a single administration of reFSH may be as good, if not better, than twice daily administration. The number of follicles and ovulations per ovulated mare were relatively high, especially considering that control mares showed no follicular activity during the treatment period. On average the number of follicles that reached ≥35 mm in diameter was 5.2 and the number of ovulations was 5.0. Similarly, two previous studies carried out on deep anestrous mares given 0.65 mg of reFSH, the average number of follicles and ovulations were 4.0 and 3.6, respectively [21,22]. Moreover, the ovulations induced in deepanestrous mares resulted in viable embryos, although the number of embryos recovered was considerably lower than the number of ovulations. The average number of embryos recovered per ovulated mare is comparable to the embryo recovery found in mares after treatment with partially purified eFSH during the transition period in two studies by Raz and coworkers (average: 2.2) [17,28]. Studies conducted during the breeding season also observed a similar number of follicles, ovulations and embryos recovered after treatment with reFSH [20,29]. Embryo recovery rates/ovulated mare in this study ranged from 75-100% depending on the dose of reFSH, which is higher than what is generally reported for nonstimulated cycles, ranging from 50% to 70% embryo recovery per cycle [30]. Similarly, treatment with eFSH (Bioniche) resulted in a 90% embryo recovery rate/cycle compared to 42% in mares receiving no treatment [31]. However, embryo recovery rates per ovulation, which in this study ranged from 37-47%, are generally lower in superovulated mares compared to non-stimulated mares. Logan and coworkers (2007) reported an embryo recovery rate per ovulation in cycling mares that was lower after treatment with
Journal Pre-proof
eFSH than in controls [32]. Speculation has it that the ovulation fossa prevents multiple oocytes from escaping the ovary into the oviduct [33]. In this study, multiple mares produced 4-5 embryos in a single flush (n=6 mares), which indicates that mares are capable of ovulating at least two fertile oocytes per ovary. The embryo recovery rates were high (75%) for mares ovulating 5-6 follicles, which requires at least one ovary undergoing a minimum of 3 ovulations. On the other hand, mares with more than 6 ovulations presented greatly decreased embryo recovery rates, suggesting that more than 3 ovulations per ovary may be detrimental to oocyte deposition into the oviduct, oocyte quality, fertilization rates, or embryo development. A study by Carmo and coworkers [34] indicated that the number of oocytes collected surgically from the oviducts of superovulated mares with EPE was higher than from control mares. The number of oocytes collected per ovulation was greater for EPE-treated mares with 1-3 ovulations than for EPE-treated mares with more than three ovulations. The oocyte recovery rate in EPE-treated mares with three or fewer ovulations was the same as in untreated control mares. The authors found that the ovulation fossa in the superovulated mares had large amounts of coagulated blood which was not observed in the control mares, and concluded that excessive hemorrhage in multiple-ovulating ovaries could be involved in the failure of the oocytes to enter the oviduct. Further refining of the protocol may increase embryo recovery rates. It is interesting to note that when LH was added to the treatment, there was an increase in embryo recovery per ovulation. Meyers-Brown reported that recovery after treatment with reFSH/reLH was 83% compared to 66% in cycling mares treated with reFSH alone [29]. The effect of LH on the ovulation fossa is unknown but it may enhance oocyte development and maturation [29]. Some of the variations in recovery
Journal Pre-proof
rates may be a function of whether the ovulations were from one ovary or both. Bilateral ovulations may prove to be a better condition for the escape of oocytes through both ovulations fosses resulting in higher embryo recovery per ovulation. The effect of multiple follicular development and multiple ovulations on fertilization rate, oocyte quality and early embryo development has not been addressed, but given the altered hormonal environment resulting from multiple follicles/corpus lutea it is possible that these events may be affected. Pregnancy rates in recipient mares are also quite variable depending on the study. In a large study carried out by Jacob and coworkers (2012), in which donor mares did not receive any stimulatory agent, the average pregnancy rates for recipient mares (nonsurgical transfer) between days +1 to -3 was 69% [35]. The average pregnancy rate for recipient mares using the Wilsher technique (vaginal speculum and cervical grasping forceps) was 92.3% compared to 70.9% using the conventional technique [4]. Recipient pregnancy rate per non-surgically transferred embryo in mares that received eFSH was 33% compared to 67% in control mares [36]. Pregnancy rates in the present study were 56-75% depending on the dose of reFSH received. Both recovery rates and pregnancy rates were quite acceptable considering the time of year this study was carried out. The ovaries of mares in deep anestrus were inactive with ≤ 10 mm in diameter follicles prior to the start of treatment. To be able to stimulate the ovary in 6-7 days is remarkable. Additionally, 14-16 embryos were transferred from superovulated mares compared to 0 in controls.
Journal Pre-proof
5.0 Conclusion These results indicate that treatment with reFSH during deep anestrus results in follicular development, ovulation of fertile oocytes, and production of embryos that established viable pregnancies after transfer. Also, a single daily administration of reFSH was as effective as two daily administrations, which allows for a simplified administration regimen. Acknowledgements Dr Jorge McDonough† and Sebastian Bereterbide from La Irenita Embriones, Daireux, Argentina. AspenBio Pharma provided the reFSH used in this study. References [1]
Squires EL, Carnevale EM, McCue PM, Bruemmer JE. Embryo technologies in the horse. Theriogenology 2003;59:151–70. doi:10.1016/S0093-691X(02)012682.
[2]
Squires EL, McCue PM, Vanderwall D. The current status of equine embryo transfer. Theriogenology 1999;51:91–104.
[3]
Stout TAE. Equine embryo transfer: review of developing potential. Equine Vet J 2006;38:467–78. doi:10.2746/042516406778400529.
[4]
Cuervo-Arango J, Claes AN, Stout TA. Effect of embryo transfer technique on the likelihood of pregnancy in the mare: a comparison of conventional and Wilsher’s forceps-assisted transfer. Vet Rec 2018;183:323–323. doi:10.1136/vr.104808.
[5]
Panarace M, Pellegrini RO, Basualdo MO, Belé M, Ursino DA, Cisterna R, et al. First field results on the use of stallion sex-sorted semen in a large-scale embryo transfer program. Theriogenology 2014;81:520–5. doi:10.1016/j.theriogenology.2013.10.021.
[6]
Panzani D, Rota A, Marmorini P, Vannozzi I, Camillo F. Retrospective study of factors affecting multiple ovulations, embryo recovery, quality, and diameter in a commercial equine embryo transfer program. Theriogenology 2014;82:807–14. doi:10.1016/j.theriogenology.2014.06.020.
[7]
Burkhardt J. Transition from anoestrus in the mare and the effects of artificial lighting. J Agric Sci 1947;37:64–8. doi:10.1017/S0021859600013083.
[8]
Palmer E, Driancourt MA, Ortavant R. Photoperiodic stimulation of the mare
Journal Pre-proof
during winter anoestrus. J Reprod Fertil Suppl 1982;32:275–82. [9]
Scraba ST, Ginther OJ. Effects of lighting programs on onset of the ovulatory season in mares. Theriogenology 1985;24:667–79.
[10] Ginther OJ. Anovulatory season. In: Ginther OJ, editor. Reprod. Biol. Mare. Second edi, Cross Plains, WI: Equiservices; 1992, p. 136–72. [11] McCue PM, Logan NL, Magee C. Management of the transition period: Hormone therapy. Equine Vet Educ 2007;19:215–21. doi:10.2746/095777307X187793. [12] Lapin DR, Ginther OJ. Induction of ovulation and multiple ovulations in seasonally anovulatory and ovulatory mares with an equine pituitary extract. J Anim Sci 1977;44:834–42. doi:10.2527/jas1977.445834x. [13] Coy RE, McCue PM, Bruemmer JE, Squires EL. Follicular response in mares in deep or transitional anestrus to equine pituitary extract. Proc Equine Nutr Physiol Symp 1999;16:65–8. [14] Niswender KD, Mccue PM, Squires EL. Effect of Purified Equine FollicleStimulating Hormone on Follicular Development. J Equine Vet Sci 2004;24:37–9. doi:10.1016/jevs.2003.12.011. [15] Peres KR, Fernandes CB, Alvarenga MA, Landim-Alvarenga FC. Effect of eFSH on Ovarian Cyclicity and Embryo Production of Mares in Spring Transitional Phase. J Equine Vet Sci 2007;27:176–80. doi:10.1016/j.jevs.2007.02.009. [16] Raz T, Hunter B, Carley S, Card C. Reproductive performance of donor mares subsequent to eFSH treatment in early vernal transition: Comparison between the first, second, and mid-season estrous cycles of the breeding season. Anim Reprod Sci 2009;116:107–18. doi:10.1016/j.anireprosci.2008.12.008. [17] Raz T, Amorim MD, Stover BC, Card CE. Ovulation, pregnancy rate and early embryonic development in vernal transitional mares treated with equine- or porcine-FSH. Reprod Domest Anim 2010;45:287–94. doi:10.1111/j.14390531.2008.01296.x. [18] Woods GL, Ginther OJ. Ovarian response, pregnancy rate, and incidence of multiple fetuses in mares treated with an equine pituitary extract. J Reprod Fertil Suppl 1982;32:415–21. [19] Woods GL, Ginther OJ. Intrauterine embryo reduction in the mare. Theriogenology 1983;20:699–706. [20] Meyers-Brown GA, McCue PM, Niswender KD, Squires EL, DeLuca CA, Bidstrup LA, et al. Superovulation in Mares Using Recombinant Equine Follicle Stimulating Hormone: Ovulation Rates, Embryo Retrieval, and Hormone Profiles. J Equine Vet Sci 2010;30:560–8. doi:10.1016/j.jevs.2010.09.007. [21] Meyers-Brown GA, McCue PM, Troedsson MHT, Klein C, Zent W, Ferris RA, et al. Induction of ovulation in seasonally anestrous mares under ambient lights using recombinant equine FSH (reFSH). Theriogenology 2013;80:456–62.
Journal Pre-proof
doi:10.1016/j.theriogenology.2013.04.029. [22] Meyers-Brown GA, Loud MC, Hyland JC, Roser JF. Deep anestrous mares under natural photoperiod treated with recombinant equine FSH (reFSH) and LH (reLH) have fertile ovulations and become pregnant. Theriogenology 2017;98:108–15. doi:10.1016/j.theriogenology.2017.05.001. [23] Jennings MW, Boime I, Daphna-Iken D, Jablonka-Shariff A, Conley AJ, Colgin M, et al. The efficacy of recombinant equine follicle stimulating hormone (reFSH) to promote follicular growth in mares using a follicular suppression model. Anim Reprod Sci 2009;116:291–307. doi:10.1016/j.anireprosci.2009.01.013. [24] Jablonka-Shariff A, Roser JF, Bousfield GR, Wolfe MW, Sibley LE, Colgin M, et al. Expression and bioactivity of a single chain recombinant equine luteinizing hormone (reLH). Theriogenology 2007;67:311–20. doi:10.1016/j.theriogenology.2006.06.013. [25] McKinnon AO, Squires EL. Morphological assessment of the equine embryo. J Am Vet Med Assoc 1988;192:401–6. [26] Douglas RH, Nuti L, Ginther OJ. Induction of ovulation and multiple ovulation in seasonally-anovulatory mares with equine pituitary fractions. Theriogenology 1974;2:133–42. doi:10.1016/0093-691X(74)90063-6. [27] Raz T, Gray A, Hunter B, Card C. Early effects of equine FSH (eFSH) treatment on hormonal and reproductive parameters in mares intended to carry their own pregnancy. Anim Reprod Sci 2009;115:76–87. doi:10.1016/j.anireprosci.2008.11.001. [28] Raz T, Carley S, Card C. Comparison of the effects of eFSH and deslorelin treatment regimes on ovarian stimulation and embryo production of donor mares in early vernal transition. Theriogenology 2009;71:1358–66. doi:10.1016/j.theriogenology.2008.09.048. [29] 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;128:52–9. doi:10.1016/j.anireprosci.2011.09.002. [30] Squires EL. Breakthroughs in Equine Embryo Cryopreservation. Vet Clin North Am - Equine Pract 2016;32:415–24. doi:10.1016/j.cveq.2016.07.009. [31] Niswender KD, Alvarenga MA, McCue PM, Hardy QP, Squires EL. Superovulation in Cycling Mares Using Equine Follicle Stimulating Hormone (eFSH). J Equine Vet Sci 2003;23:497–500. doi:10.1016/j.jevs.2003.10.002. [32] Logan NL, McCue PM, Alonso MA, Squires EL. Evaluation of three equine FSH superovulation protocols in mares. Anim Reprod Sci 2007;102:48–55. doi:10.1016/j.anireprosci.2006.09.027. [33] Allen W. The Development and Application of the Modern Reproductive Technologies to Horse Breeding. Reprod Domest Anim 2005;40:310–29.
Journal Pre-proof
doi:10.1111/j.1439-0531.2005.00602.x. [34] Carmo MT, Losinno L, Aquilar JJ, Araujo GHM. Oocyte transport to the oviduct of superovulated mares. Anim Reprod Sci 2006;94:337–9. doi:10.1016/j.anireprosci.2006.03.080. [35] 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;77:1159–66. doi:10.1016/j.theriogenology.2011.10.022. [36] Raz T, Green G, Carley S, Card C. Folliculogenesis, embryo parameters and post-transfer recipient pregnancy rate following equine follicle-stimulating hormone (eFSH) treatment in cycling donor mares. Aust Vet J 2011;89:138–42. doi:10.1111/j.1751-0813.2011.00691.x.
Journal Pre-proof
Highlights
reFSH stimulates follicular development and ovulation in deep anestrus mares. Once-a-day reFSH administration is sufficient for inducing superovulation. Embryos from reFSH-treated mares establish pregnancy after embryo transfer.
Journal Pre-proof
Table 1. Ovulation and embryo recovery rates of anovulatory mares treated with reFSH Dose of 0.65 mg BID 1.3 mg SID reFSH Days of 5.9 ± 0.6a 7.1 ± 0.8a treatment Ovulating 8/10 8/10 mares Follicles > 35 mm in 5.2 ± 0.9a 6.4 ± 0.8a diameter Ovulations per ovulated 5.1 ± 1.0 5.5 ± 0.8 mare Embryos recovered 1.9 ± 0.5 2.6 ± 0.5 per ovulated mare Embryo recovery 75% 88% rate per (7/8) (6/8) ovulated mare Embryo recovery 37% 47% rate per ovulation Embryos 14 16 transferred Pregnancy rate per 57% 56% transferred embryo Values indicate mean ± SEM a, b: Different letters indicate p < 0.05
0.32 mg BID
Saline control
7.3 ± 0.7a
10 ± 0b
5/10
0/10
4.1 ± 1.0a
0 ± 0b
4.4 ± 0.9
-
2.0 ± 0.4
-
100% (5/5)
-
45%
-
4
-
75%
-