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Alternatives to improve a prostaglandin-based protocol for timed artificial insemination in sheep
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Theriogenology 76 (2011) 1501–1507 www.theriojournal.com
Alternatives to improve a prostaglandin-based protocol for timed artificial insemination in sheep J. Olivera-Muzantea,*, J. Gilb, S. Fierroa, A. Menchacac, E. Rubianesd a
Departamento de Salud en los Sistemas Pecuarios, Área de Producción y Sanidad Ovina, Facultad de Veterinaria, EEMAC. Ruta 3 Km 363. 60000 PO Box 57072 Paysandú, Uruguay b Departamento de Salud en los Sistemas Pecuarios, Área de Teriogenología, Facultad de Veterinaria, EEMAC. Ruta 3 Km. 363. 60000 PO Box 57072. Paysandú, Uruguay c Instituto de Reproducción Animal Uruguay, Fundación IRAUy, Camino Cruz del Sur 2250, 12200, Montevideo, Uruguay d Departamento de Producción Animal y Pasturas, Facultad de Agronomía, Garzón 780, 12900 Uruguay Received 22 April 2011; received in revised form 14 June 2011; accepted 17 June 2011
Abstract The objective was to improve the reproductive performance of a prostaglandin (PG) F2␣-based protocol for timed artificial insemination (TAI) in sheep (Synchrovine®: two doses of 160 g of delprostenate 7 d apart, with TAI 42 h after second dose). Three experiments were performed: Experiment 1) two doses of a PGF2␣ analogue (delprostenate 80 or 160 g) given 7 d apart; Experiment 2) two PGF2␣ treatment intervals (7 or 8 d apart) and two times of TAI (42 or 48 h); and Experiment 3) insemination 12 h after estrus detection or TAI with concurrent GnRH. Experiments involved 1131 ewes that received cervical insemination with fresh semen during the breeding season (32/34 °S–58 °W). Estrous behaviour, conception rate, prolificacy, and fecundity (ultrasonography 30–40 d), were assessed. In Experiment 1, ewes showing estrus between 25 and 48 h or at 72 h after the second PGF2␣ did not differ between 80 and 160 g of delprostenate (73 vs 86%, P ⫽ 0.07; and 92 vs 95%, P ⫽ NS, respectively). Conception rate and fecundity were lower (P ⬍ 0.05) using 80 vs 160 g (0.24 vs 0.42, and 0.27 vs 0.47, respectively). In Experiment 2, giving PGF2␣ 7 d apart resulted in higher (P ⬍ 0.05) rates of conception (0.45 and 0.51) and fecundity (0.49 and 0.53) than treatments 8 d apart (conception: 0.33 and 0.29; fecundity: 0.33 and 0.34) for TAI at 42 and 48 h, respectively. In Experiment 3, rates of conception, prolificacy and fecundity were similar (NS) between Synchrovine® with TAI at 42 h (0.50, 1.13, and 0.56) and AI 12 h after estrus detection (0.47, 1.18, and 0.55), and Synchrovine® plus GnRH at TAI (0.38, 1.28, and 0.49). However, all TAI treatments had lower (P ⬍ 0.05) prolificacy and fecundity compared to AI following detection of spontaneous estrus (1.39 and 0.83, respectively). In conclusion, the Synchrovine® protocol was: a) more successful using 160 vs 80 g delprostenate; b) more successful with a 7 d than 8 d PGF2␣ interval; c) similarly effective for TAI versus AI 12 h after estrus detection; and d) not improved by giving GnRH at TAI. © 2011 Elsevier Inc. All rights reserved. Keywords: Ewes; Estrus synchronization; AI; Conception
1. Introduction Timed artificial insemination (TAI) is an important tool when estrus detection is not feasible. It provides synchronized inseminations and more efficient use of * Corresponding author. Tel.: ⫹⫹598 47241282; fax: ⫹⫹598 47227950. E-mail address: [email protected] (J. Olivera-Muzante). 0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.06.020
superior males [1]. Currently, widespread application of these biotechnologies under commercial field conditions requires easy implementation procedures, acceptable pregnancy rates, and low environmental impact [2]. Conventional TAI protocols involve intravaginal devices impregnated with progestins, in conjunction with eCG, yielding acceptable pregnancy rates both within and outside the physiological breeding season [3,4]. Nevertheless, repeated use of eCG has been as-
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sociated with a humoral immune response in ewes and goats [5,6] and development of ovarian follicular cysts [7], followed by low pregnancy rates. In addition, use of a progestin and eCG has been limited in some countries due to public health concerns [8] and animal welfare issues [9]. However, prostaglandin-based protocols may be a viable alternative to the use of progestagens. Conventional PGF2␣ treatments consist of two doses given 9 to 12 d apart [4]. Using this protocol, a high proportion of ewes are detected in estrus after the second dose, but over a 4 –5 d interval [10,11], making estrus detection necessary and rendering this protocol inappropriate for TAI. Conversely, Rubianes et al [12] and Contreras-Solis et al [13] demonstrated that the refractoriness of a recently formed ovine CL to PGF2␣ might be restricted to the first 2 d after ovulation, with a consistent interval from treatment to ovulation (60.8 ⫾ 1.8 or 61.1 ⫾ 1.1 h, respectively). Based on these observations, a protocol to synchronize estrus and ovulations (Synchrovine®, MIEM - Cámara Nacional de Registros, Montevideo Uruguay), with two large doses of PGF2␣ given 7 d apart was proposed [1]. Synchrovine® promotes highly synchronous estrus during the first 72 h, with 80% of the ewes in estrus within 25 to 48 h after the second PGF2␣ dose, making it practical for use in TAI programs. However, this protocol yielded lower conception rates after cervical or intrauterine TAI compared to the conventional P4-eCG protocol [14] or spontaneous estrus [15]. Thus, other alternatives to improve this protocol need to be studied. A substantive reduction of the cloprostenol dose was effective in inducing luteolysis, estrus onset, ovulation, and subsequent development of normal CLs [16,17]. The reduced dose might decrease side effects, including changes in cervical mucus and uterine contractions [18,19]. A longer interval between PGF2␣ treatments increased the sensitivity of young CLs to this hormone [20] and increased progesterone concentrations during development of the designated preovulatory follicle. When feasible, AI associated with estrus detection may be used to improve Synchrovine® results. However, a protocol including GnRH treatment 36 h after PGF2␣ resulted in an LH surge, ovulation within 48 h, and a fully functional CL [21]. Therefore, administration of GnRH at AI would be a practical option if it were to improve ovulation and fertility, as demonstrated in dairy cows [22,23]. The aims of the present study were to determine whether either a reduction of PGF2␣ luteolytic dose, an increase in the interval between PGF2␣ treatments, AI of ewes detected in estrus, or giving GnRH at TAI,
improved reproductive outcomes for a Synchrovine® synchronization protocol. 2. Materials and methods 2.1. Animals All procedures were approved by the University’s Animal Experimentation Committee (CHEA-UdelaR). The studies were done during the physiologic breeding season (March–April), involving 1131 clinically healthy, nulliparous and multiparous ewes (1.5–5.5 y old), with good body condition (score 3.4 ⫾ 0.4, mean ⫾ SD [24]), grazing natural pastures at three locations (Experiment 1: “El Coraje” Farm, Lavalleja Uruguay, 34 °S/56’ W; Experiment 2: “Piedra Mora” Farm, Paysandú Uruguay, 32 °S/ 57’ W; and Experiment 3: “Mario A. Cassinoni” Experimental Station, Paysandú Uruguay, 32 °S/ 58’ W). On these farms, 13 clinically healthy rams aged 1.5–5.5 y old, grazing natural improved pastures (with lotus and rye grass) were used as semen donors. 2.2. Experiments Three experiments were done. Ewes and rams were either randomly assigned to treatments (Experiment 1 and 2) or allocated to treatments, on the basis of breed, parity, and body condition score (Experiment 3). 2.2.1. Experiment 1 Corriedale multiparous ewes (n ⫽ 127) were subjected to cervical TAI (April 12 and 13, Year 1) with fresh semen from two Corriedale rams. Two PGF2␣ doses for Synchrovine® were compared: ● Synchrovine®: Two doses of delprostenate (160 g im; Glandinex®, Universal Lab, Montevideo, Uruguay) given 7 d apart, with TAI 42 h after the second dose (Control). ● Synchrovine®-LD: As above, but using 80 g delprostenate. 2.2.2. Experiment 2 Australian Merino multiparous ewes (n ⫽ 583) were cervically TAI (April 11 and 12, Year 2), with fresh extended semen from five rams (three Australian Merino and two Suffolk). In a factorial design, two PGF2␣ treatment intervals (7 or 8 d) and two AI-times (42 or 48 ⫾ 1 h after the second dose of PGF2␣) for Synchrovine® were compared. ● Synchrovine®: as described in Experiment 1. ● Synchrovine®-48: as above, with TAI at 48 h after second dose.
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● Synchrovine®8-42: two doses of PGF2␣ 8 d apart, with TAI 42 h after the second dose. ● Synchrovine®8-48: two doses of PGF2␣ 8 d apart, with TAI 48 h after the second dose. 2.2.3. Experiment 3 Four hundred and twenty one ewes (345 multiparous and 76 nulliparous) Corriedale and crosses (x Texel, x Milchschaf, or x Ile de France; n ⫽ 209, 58, 74 and 80, respectively) were cervically AI (March 18 to 21, Year 1) with fresh semen from six rams (two Corriedale, three Southdown and one Poll Dorset). Four treatments were compared: ● Synchrovine®: as described in Experiment 1. ● Synchrovine®-DE: Synchrovine®, with AI 12 h after detected estrus. ● Synchrovine®-GnRH: Synchrovine® plus GnRH (busereline acetate 8 g im; Receptal®, Intervet International GmbH, Unterscheleiheim, Germany) at TAI, 42 h after the second PGF2␣. ● Spontaneous Estrus: ewes were pre-synchronized with one dose of PGF2␣ (delprostenate 160 g im) 17 d in advance, ignoring the induced estrus, with AI 12 h after detection of the following spontaneous estrus. 2.3. Semen processing Semen was collected with an artificial vagina and estrous teaser ewes. All rams delivered ejaculates with acceptable volume (0.75–2 mL), concentration (⬎ 3.0 ⫻ 109 sperm/mL), total motility (⬎ 70%), and sperm morphology (⬍ 10% total sperm abnormalities). During the experiment, two consecutive ejaculates from each ram were collected within 10 min, pooled, and treated as a single ejaculate. Ejaculates from each ram were placed in a portable water bath at 33 °C and subjectively assessed (motility, volume and color). Sperm concentration was assessed with a photometer (Spermacue®, Minitub, Landshut, Germany). Fresh undiluted AI doses (200 ⫻ 106 sperm/ ewe; Experiments 1 and 3) or extended in UHT skim milk with antibiotics (final concentration 1000 ⫻ 106 sperm/mL, insemination dose 0.2 mL/ewe; Experiment 2) were maintained at 30 °C and used within 30 min after collection. 2.4. Estrus detection and AI The onset of estrus was determined twice daily during the first 72 h after the second PGF2␣ treatment in ewes in Experiment 1 (Synchrovine® and Synchrovine®-LD), and Experiment 3 (Synchrovine®-
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DE), or 17 d after PGF2␣ dose in Experiment 3 (Control, spontaneous estrus). Five vasectomized rams/100 ewes (Experiment 1) or 10 androgenized wethers/100 ewes (Experiment 3) were used as painted teaser animals. Wethers were treated with 100 mg testosterone cyclopentylpropionate im (Testosterona Ultra Fuerte®, Laboratorios Dispert, Montevideo, Uruguay), given on three occasions (14, 7 and 1 d, respectively, before the beginning of the estrus detection period). Paint was applied twice daily. Cervical AI was done using a speculum equipped with a light source and an insemination gun (Walmur Veterinary Instruments, Montevideo, Uruguay), slowly depositing the semen as deep as possible into the cervix. 2.5. Reproductive performance Estrous behaviour (ewes in estrus/treated) and its distribution within 72 h after the second PGF2␣ treatment was assessed (Experiments 1 and 3). Rates of conception (pregnant/inseminated ewes), prolificacy (fetuses/pregnant ewe), and fecundity (fetuses/inseminated ewes), were evaluated 30 d (Experiments 1 and 3) or 40 d (Experiment 2) after TAI by transrectal or transabdominal ultrasonography (Aloka® 500, Tokyo, Japan; 5.0 MHz linear-array or 3.5 MHz convex-array, respectively). 2.6. Statistical analyses Differences in estrous behaviour and its distribution, and rates of conception, prolificacy and fecundity among synchronization protocols or spontaneous estrus after AI were analyzed by ANOVA for categorical variables (PROC CATMOD; SAS). Data are presented as means, with P ⬍ 0.05 considered significantly different. 3. Results 3.1. Experiment 1 The results are shown in Fig. 1 and Table 1. The PGF2␣ dose did not significantly affect the proportion of ewes coming into estrus up to 72 h after the second PGF2␣ (95 vs 92%; P ⫽ NS), with a high concentration between 25 and 48 h (86 vs 73%, P ⫽ 0.07) for Synchrovine® and Synchrovine®-LD respectively. However, reproductive performance was better (P ⬍ 0.05) in Synchrovine® treated ewes compared to Synchrovine®-LD.
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Fig. 1. Distribution of estrous ewes treated with two doses ( □ Synchrovine®-LD, 80 g; Synchrovine®, 160 g) of PGF2␣ (delprostenate) given 7 d apart (n⫽127; Experiment 1). There were no significant differences between experimental groups.
3.2. Experiment 2 Reproductive performance (conception, prolificacy, and fecundity) for each group is shown (Table 2). There was no AI-time effect (42 vs 48 h, P ⫽ NS) within the PGF2␣ treatment interval (7 or 8 d apart). Nevertheless, there was a PGF2␣ treatment interval effect, with better reproductive performance (P ⬍ 0.05) in ewes given PGF2␣ 7 d apart compared to those treated 8 d apart. The interaction PGF2␣ treatment interval*AI time was significant (P ⬍ 0.05) just for prolificacy, but not for conception or final fecundity (P ⫽ NS). 3.3. Experiment 3 The proportion of ewes showing estrus between 25 and 48 h and the total number showing estrus by 72 h after the second PGF2␣ in Synchrovine®:DE group were 81 and 92%, respectively. Neither estrus detection
Table 1 Reproductive responses obtained with two doses of PGF2␣ (delprostenate) and cervical insemination with fresh semen in sheep (Experiment 1). Conception Synchrovine® (n ⫽ 64) Synchrovine®-LD (n ⫽ 63)
Prolificacy
Fecundity
0.42 (27/64)
1.11 (30/27)
0.47a (30/64)
0.24b (15/63)
1.13 (17/15)
0.27b (17/63)
a
Synchrovine®, two PGF2␣ treatments 7 d apart (160 g delprostenate) and TAI 42 h after second PGF2␣; Synchrovine®-LD, two PGF2␣ treatments 7 d apart (80 g delprostenate) and TAI 42 h after second PGF2␣; Conception, pregnant at 30 d after AI/inseminated ewes; Prolificacy, fetuses/pregnant ewe; Fecundity, fetuses/inseminated ewe. a,b Within a column, rates without a common superscript differed (P ⬍ 0.05).
(Synchrovine®-DE) nor GnRH at TAI (Synchrovine®GnRH) significantly improved (P⫽NS) the reproductive performance of synchronized ewes. All groups subjected to these synchronization protocols had lower (P ⬍ 0.05) fecundity than the Control group (spontaneous estrus, Table 3). 4. Discussion In the present study, both the amount of PGF2␣ and the interval between PGF2␣ treatments significantly affected reproductive performance of synchronized ewes. However, neither estrus detection (to determine time of AI) nor administration of GnRH at TAI significantly affected the reproductive outcome of the Synchrovine® protocol. The cumulative proportion of ewes showing estrus by 72 h after the second PGF2␣ treatment was similar between the full and half dose of PGF2␣ analog (Experiment 1), but the ewes in estrus between 25 and 48 h in the group treated with the full dose (Synchrovine®) tended to be higher, with a sharp onset of estrus. This could account for the better reproductive performance in this group. These results contradicted other basic experiments using a reduced dose of cloprostenol [16,17], but agreed with other studies [20] regarding estrus response following dinoprost tromethamine treatment (10 vs 5 mg) of young CLs (3.5 to 5 d old). The concept that a reduced dose of PGF2␣ would improve the reproductive performance, based on reduced side effects, was not supported. Perhaps some of the young CLs failed to respond to PGF2␣ [25], as observed in the Synchrovine® protocol, thereby reducing the number of ewes that were close to the time of ovulation at TAI.
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Table 2 Reproductive performance in sheep obtained with two PGF2␣ (delprostenate) at two intervals and cervical insemination (fresh semen) at two times (Experiment 2). Conception Synchrovine® (n ⫽ 145) Synchrovine®-48 (n ⫽ 145) Synchrovine®8-42 (n ⫽ 147) Synchrovine®8-48 (n ⫽ 146)
a
0.45 (65/145) 0.51a (74/145) 0.33b (48/147) 0.29b (42/146)
Prolificacy a
1.09 (71/65) 1.04a (77/74) 1.02a (49/48) 1.19b (50/42)
Fecundity 0.49a (71/145) 0.53a (77/145) 0.33b (49/147) 0.34b (50/146)
Synchrovine®, two PGF2␣ treatments 7 d apart and TAI 42 h after second PGF2␣; Synchrovine®-48, two PGF2␣ treatments 7 d apart and TAI 48 h after second PGF2␣; Synchrovine®8-42, two PGF2␣ treatments 8 d apart and TAI 42 h after second PGF2␣; Synchrovine®8-48, two PGF2␣ treatments 8 d apart and TAI 48 h after second PGF2␣; Conception, pregnant at 40 d after AI/inseminated ewes; Prolificacy: fetuses/pregnant ewe; Fecundity, fetuses/inseminated ewe. a,b Within a column, rates without a common superscript differed (P ⬍ 0.05).
Variations in the time of insemination after the last PGF2␣ had no significant effects (Experiment 2). As observed previously with this TAI protocol using fresh semen, pregnancy rates were similar after cervical inseminations both at 42 and 48 h [14,26], offering a reasonable time-window for use of this protocol in the field. However, reproductive performance was significantly affected by altering the interval between doses of PGF2␣ in the Synchrovine® protocol, with better results for treatments given 7 vs 8 d apart. Although estrus response and ovulation was not determined in this experiment, as in a previous study by Rubianes et al [12], we speculate that the interval between treatment and ovulation was more varied in ewes treated 8 d apart. This was probably due to a higher proportion of ovulations coming from the second follicular wave, resulting in TAI occurring too early for optimal fertility. Experiment 3 tested the effect of estrus detection, with only ewes in estrus inseminated, and GnRH at TAI. Firstly, AI of ewes showing estrus after PGF2␣ treatment did not improve the conception rate of this new synchronization protocol using fresh semen. The higher cumulative proportion of ewes that showed es-
trus between 25 and 48 h after the second PGF2␣ in the Synchrovine®-DE group, and the prolonged lifespan of fresh semen, may explain the similar conception rate achieved in the Synchrovine® TAI group. Therefore, we concluded that estrus detection is not required when using this protocol with fresh semen, reinforcing the advantage of TAI procedures. Secondly, giving a GnRH analogue concurrent with TAI did not improve the final reproductive outcomes of Synchrovine® protocol. Furthermore, the conception rate of this treatment tended to be lower (P ⫽ 0.09), but the prolificacy was higher (P ⫽ 0.07) than in the Synchrovine® group. These results agreed with previous reports where, despite the synchrony in ovulation achieved, giving GnRH within 24 to 44 h after a P4-eCG treatment failed to improve ewe fertility [27–29]. Other authors reported a tendency to increase litter size as a result of GnRH treatment immediately after the AI of ewes synchronized with a P4-eCG-PGF2␣ [30], or 30 h after a P4-PGF2␣ or P4-PGF2␣-eCG treatment [8]; this was attributed to GnRH increasing ovulation rates [30 –32]. In another study, the male effect was used after a short-interval, cloprostenol-based protocol to induce an earlier preovulatory LH surge and ovulation, thus im-
Table 3 Reproductive response obtained with variations of Synchrovine® protocol using cervical insemination with fresh semen in sheep (Experiment 3). Conception Synchrovine® (n ⫽ 111) Synchrovine®-DE (n ⫽ 102) Synchrovine®-GnRH (n ⫽ 104) Spontaneous Estrus (Control, n ⫽ 104)
ab
0.50 (55/111) 0.47ab (44/94) 0.38a (40/104) 0.59b (51/86)
Prolificacy a
1.13 (62/55) 1.18a (52/44) 1.28ab(51/40) 1.39b (71/51)
Fecundity 0.56a (62/111) 0.55a (52/94) 0.49a (51/104) 0.83b (71/86)
Synchrovine®, two PGF2␣ treatments 7 d apart and TAI 42 h after second PGF2␣; Synchrovine®-DE, two PGF2␣ treatments 7 d apart and AI 12 h after detected estrus; Synchrovine®-GnRH, two PGF2␣ treatments 7 d apart plus 8 g GnRH (busereline acetate) at TAI (42 h after second PGF2␣); Spontaneous Estrus, pre synchronized ewes with one dose of PGF2␣ 17 d in advance, ignoring the induced estrus, and AI 12 h after detected spontaneous estrus; Conception, pregnant 30 d after AI/inseminated ewes; Prolificacy, fetuses/pregnant ewe; Fecundity, fetuses/ inseminated ewe. a,b Within a column, rates without a common superscript differed (P ⬍ 0.05).
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proving fertility compared to a progesterone-based protocol [13]. Consequently, a GnRH dose was expected to favor the Synchrovine® TAI protocol. Assuming that ovulation in Synchrovine® protocol occurs approximately 60 h after the second PGF2␣ treatment [12,13], and that the physiological LH-surge is ⬃24 h before ovulation [33], the LH surge should be ⬃36 h after the last PGF2␣. If the GnRH was given at TAI (42 h), perhaps this promoted a second LH surge 2– 4 h later [21,28]. In this regard, the GnRH was probably too late to augment the endogenous LH surge to target a timed ovulation. Although it is logistically easier to give GnRH concurrent with TAI, earlier administration should be considered in future trials. Finally, the Synchrovine® protocol and the studied alternatives had lower reproductive outcomes compared to spontaneous estrus and AI based on estrus detection. An environment dominated by lower progesterone concentration during the growth phase of the ovulatory follicle, stimulating a faster growth rate, and a larger follicular size, appeared to be a cause of the lower ovulation rate, conception, and prolificacy achieved with Synchrovine® protocol compared to ewes AI in spontaneous estrus [15]. In conclusion, reproductive performance of the Synchrovine® protocol was: a) better when using 160 versus 80 g delprostenate; b) better when using a 7 d interval compared to an 8 d interval between PGF2␣ treatments; c) similar with insemination at prefixed time or AI 12 h after estrus detection; and d) not improved by giving GnRH at TAI.
Acknowledgments This work was supported by funds from Universidad de la República (CSIC 600/6015) and MGAP-DILAVE “M.C. Rubino”. We acknowledge “Coraje” Farm (Dighiero-Triay family), “Piedra Mora” Farm (FilliolBarreiro family) and EEMAC (Facultad de Agronomía, UdelaR, Paysandú, Uruguay) for allowing us to use their facilities and animals. Thanks also to E. Campos and S. Kmaid from Universal Lab and J. Amaro from Dispert for donating delprostenate and testosterone, respectively. We are also grateful to J.L. Alabart for statistical analysis; to G. McCann for revising the manuscript; and to S. Forichi, M. Correa, A. Araujo, E. Filliol, and G. Stoletniy for their support during the trials. The authors do not have any financial or personal relationships with other people or organizations that could have inappropriately biased or influenced this work.
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J. Olivera-Muzante et al. / Theriogenology 76 (2011) 1501–1507 [16] Hearnshaw H, Restall BJ, Nancarrow CD, Mattner PE. Synchronization of oestrus in cattle, sheep and goats using a prostaglandin analogue. Proc Aust Soc Anim Prod 1974;10:242–5. [17] Contreras-Solís I, Vásquez B, Díaz T, Letelier C, López Sebastián A, González-Bulnes A. Ovarian and endocrine responses in tropical sheep treated with reduced doses of cloprostenol. Anim Reprod Sci 2009;114:384 –92. [18] Hawk HW. Uterine motility and sperm transport in the estrous ewes after prostaglandin induced regression of corpora lutea. J Anim Sci 1973;37:1380 –5. [19] Prasad A, Kalyan NK, Bachlaus NK, Arora FC, Pandley RS. Biochemical changes in the cervical mucus of buffalo after induction of oestrous with prostaglandin F2 alpha and cloprostenol. J Reprod Fert 1981;62:583–7. [20] Pope WF, Cárdenas H. Sensitivity of sheep to exogenous prostaglandin F2 early in the estrous cycle. Small Rum Res 2004; 55:245– 8. [21] Rubianes E, Beard A, Dierschke DJ, Bartlewski P, Adams GP, Rawlings NC. Endocrine and ultrasound evaluation of the response to PGF2␣ and GnRH given at different stages of the luteal phase in cyclic ewes. Theriogenology 1997;48:1093–104. [22] Lee CN, Maurice E, Ax RL, Pennington JA, Hoffman WF, Brown MD. Efficacy of gonadotrophin-releasing hormone administration at the time of artificial insemination of heifers and postpartum and repeat breeder dairy cows. Am J Vet Res 1983; 44:2160 –3. [23] Nakao T, Narila S, Tanaka K, Horn H, Shirakawa J, Noshiro H, Saga N, Tsunoda H, Kawata K. Improvement of the first-service pregnancy rate in cows with gonadotrophin-releasing hormone analog. Theriogenology 1983;20:111–9. [24] Russell AJF, Doney JM, Jun RG. Subjective assessment of body fat in live sheep. J Agric Sci 1969;72:451– 4. [25] Silva PJ, Juengel JL, Rollyson MK, Niswender GD. Prostaglandin metabolism in the ovine corpus luteum: catabolism of pros-
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Theriogenology 76 (2011) 1501–1507 www.theriojournal.com
Alternatives to improve a prostaglandin-based protocol for timed artificial insemination in sheep J. Olivera-Muzantea,*, J. Gilb, S. Fierroa, A. Menchacac, E. Rubianesd a
Departamento de Salud en los Sistemas Pecuarios, Área de Producción y Sanidad Ovina, Facultad de Veterinaria, EEMAC. Ruta 3 Km 363. 60000 PO Box 57072 Paysandú, Uruguay b Departamento de Salud en los Sistemas Pecuarios, Área de Teriogenología, Facultad de Veterinaria, EEMAC. Ruta 3 Km. 363. 60000 PO Box 57072. Paysandú, Uruguay c Instituto de Reproducción Animal Uruguay, Fundación IRAUy, Camino Cruz del Sur 2250, 12200, Montevideo, Uruguay d Departamento de Producción Animal y Pasturas, Facultad de Agronomía, Garzón 780, 12900 Uruguay Received 22 April 2011; received in revised form 14 June 2011; accepted 17 June 2011
Abstract The objective was to improve the reproductive performance of a prostaglandin (PG) F2␣-based protocol for timed artificial insemination (TAI) in sheep (Synchrovine®: two doses of 160 g of delprostenate 7 d apart, with TAI 42 h after second dose). Three experiments were performed: Experiment 1) two doses of a PGF2␣ analogue (delprostenate 80 or 160 g) given 7 d apart; Experiment 2) two PGF2␣ treatment intervals (7 or 8 d apart) and two times of TAI (42 or 48 h); and Experiment 3) insemination 12 h after estrus detection or TAI with concurrent GnRH. Experiments involved 1131 ewes that received cervical insemination with fresh semen during the breeding season (32/34 °S–58 °W). Estrous behaviour, conception rate, prolificacy, and fecundity (ultrasonography 30–40 d), were assessed. In Experiment 1, ewes showing estrus between 25 and 48 h or at 72 h after the second PGF2␣ did not differ between 80 and 160 g of delprostenate (73 vs 86%, P ⫽ 0.07; and 92 vs 95%, P ⫽ NS, respectively). Conception rate and fecundity were lower (P ⬍ 0.05) using 80 vs 160 g (0.24 vs 0.42, and 0.27 vs 0.47, respectively). In Experiment 2, giving PGF2␣ 7 d apart resulted in higher (P ⬍ 0.05) rates of conception (0.45 and 0.51) and fecundity (0.49 and 0.53) than treatments 8 d apart (conception: 0.33 and 0.29; fecundity: 0.33 and 0.34) for TAI at 42 and 48 h, respectively. In Experiment 3, rates of conception, prolificacy and fecundity were similar (NS) between Synchrovine® with TAI at 42 h (0.50, 1.13, and 0.56) and AI 12 h after estrus detection (0.47, 1.18, and 0.55), and Synchrovine® plus GnRH at TAI (0.38, 1.28, and 0.49). However, all TAI treatments had lower (P ⬍ 0.05) prolificacy and fecundity compared to AI following detection of spontaneous estrus (1.39 and 0.83, respectively). In conclusion, the Synchrovine® protocol was: a) more successful using 160 vs 80 g delprostenate; b) more successful with a 7 d than 8 d PGF2␣ interval; c) similarly effective for TAI versus AI 12 h after estrus detection; and d) not improved by giving GnRH at TAI. © 2011 Elsevier Inc. All rights reserved. Keywords: Ewes; Estrus synchronization; AI; Conception
1. Introduction Timed artificial insemination (TAI) is an important tool when estrus detection is not feasible. It provides synchronized inseminations and more efficient use of * Corresponding author. Tel.: ⫹⫹598 47241282; fax: ⫹⫹598 47227950. E-mail address: [email protected] (J. Olivera-Muzante). 0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.06.020
superior males [1]. Currently, widespread application of these biotechnologies under commercial field conditions requires easy implementation procedures, acceptable pregnancy rates, and low environmental impact [2]. Conventional TAI protocols involve intravaginal devices impregnated with progestins, in conjunction with eCG, yielding acceptable pregnancy rates both within and outside the physiological breeding season [3,4]. Nevertheless, repeated use of eCG has been as-
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sociated with a humoral immune response in ewes and goats [5,6] and development of ovarian follicular cysts [7], followed by low pregnancy rates. In addition, use of a progestin and eCG has been limited in some countries due to public health concerns [8] and animal welfare issues [9]. However, prostaglandin-based protocols may be a viable alternative to the use of progestagens. Conventional PGF2␣ treatments consist of two doses given 9 to 12 d apart [4]. Using this protocol, a high proportion of ewes are detected in estrus after the second dose, but over a 4 –5 d interval [10,11], making estrus detection necessary and rendering this protocol inappropriate for TAI. Conversely, Rubianes et al [12] and Contreras-Solis et al [13] demonstrated that the refractoriness of a recently formed ovine CL to PGF2␣ might be restricted to the first 2 d after ovulation, with a consistent interval from treatment to ovulation (60.8 ⫾ 1.8 or 61.1 ⫾ 1.1 h, respectively). Based on these observations, a protocol to synchronize estrus and ovulations (Synchrovine®, MIEM - Cámara Nacional de Registros, Montevideo Uruguay), with two large doses of PGF2␣ given 7 d apart was proposed [1]. Synchrovine® promotes highly synchronous estrus during the first 72 h, with 80% of the ewes in estrus within 25 to 48 h after the second PGF2␣ dose, making it practical for use in TAI programs. However, this protocol yielded lower conception rates after cervical or intrauterine TAI compared to the conventional P4-eCG protocol [14] or spontaneous estrus [15]. Thus, other alternatives to improve this protocol need to be studied. A substantive reduction of the cloprostenol dose was effective in inducing luteolysis, estrus onset, ovulation, and subsequent development of normal CLs [16,17]. The reduced dose might decrease side effects, including changes in cervical mucus and uterine contractions [18,19]. A longer interval between PGF2␣ treatments increased the sensitivity of young CLs to this hormone [20] and increased progesterone concentrations during development of the designated preovulatory follicle. When feasible, AI associated with estrus detection may be used to improve Synchrovine® results. However, a protocol including GnRH treatment 36 h after PGF2␣ resulted in an LH surge, ovulation within 48 h, and a fully functional CL [21]. Therefore, administration of GnRH at AI would be a practical option if it were to improve ovulation and fertility, as demonstrated in dairy cows [22,23]. The aims of the present study were to determine whether either a reduction of PGF2␣ luteolytic dose, an increase in the interval between PGF2␣ treatments, AI of ewes detected in estrus, or giving GnRH at TAI,
improved reproductive outcomes for a Synchrovine® synchronization protocol. 2. Materials and methods 2.1. Animals All procedures were approved by the University’s Animal Experimentation Committee (CHEA-UdelaR). The studies were done during the physiologic breeding season (March–April), involving 1131 clinically healthy, nulliparous and multiparous ewes (1.5–5.5 y old), with good body condition (score 3.4 ⫾ 0.4, mean ⫾ SD [24]), grazing natural pastures at three locations (Experiment 1: “El Coraje” Farm, Lavalleja Uruguay, 34 °S/56’ W; Experiment 2: “Piedra Mora” Farm, Paysandú Uruguay, 32 °S/ 57’ W; and Experiment 3: “Mario A. Cassinoni” Experimental Station, Paysandú Uruguay, 32 °S/ 58’ W). On these farms, 13 clinically healthy rams aged 1.5–5.5 y old, grazing natural improved pastures (with lotus and rye grass) were used as semen donors. 2.2. Experiments Three experiments were done. Ewes and rams were either randomly assigned to treatments (Experiment 1 and 2) or allocated to treatments, on the basis of breed, parity, and body condition score (Experiment 3). 2.2.1. Experiment 1 Corriedale multiparous ewes (n ⫽ 127) were subjected to cervical TAI (April 12 and 13, Year 1) with fresh semen from two Corriedale rams. Two PGF2␣ doses for Synchrovine® were compared: ● Synchrovine®: Two doses of delprostenate (160 g im; Glandinex®, Universal Lab, Montevideo, Uruguay) given 7 d apart, with TAI 42 h after the second dose (Control). ● Synchrovine®-LD: As above, but using 80 g delprostenate. 2.2.2. Experiment 2 Australian Merino multiparous ewes (n ⫽ 583) were cervically TAI (April 11 and 12, Year 2), with fresh extended semen from five rams (three Australian Merino and two Suffolk). In a factorial design, two PGF2␣ treatment intervals (7 or 8 d) and two AI-times (42 or 48 ⫾ 1 h after the second dose of PGF2␣) for Synchrovine® were compared. ● Synchrovine®: as described in Experiment 1. ● Synchrovine®-48: as above, with TAI at 48 h after second dose.
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● Synchrovine®8-42: two doses of PGF2␣ 8 d apart, with TAI 42 h after the second dose. ● Synchrovine®8-48: two doses of PGF2␣ 8 d apart, with TAI 48 h after the second dose. 2.2.3. Experiment 3 Four hundred and twenty one ewes (345 multiparous and 76 nulliparous) Corriedale and crosses (x Texel, x Milchschaf, or x Ile de France; n ⫽ 209, 58, 74 and 80, respectively) were cervically AI (March 18 to 21, Year 1) with fresh semen from six rams (two Corriedale, three Southdown and one Poll Dorset). Four treatments were compared: ● Synchrovine®: as described in Experiment 1. ● Synchrovine®-DE: Synchrovine®, with AI 12 h after detected estrus. ● Synchrovine®-GnRH: Synchrovine® plus GnRH (busereline acetate 8 g im; Receptal®, Intervet International GmbH, Unterscheleiheim, Germany) at TAI, 42 h after the second PGF2␣. ● Spontaneous Estrus: ewes were pre-synchronized with one dose of PGF2␣ (delprostenate 160 g im) 17 d in advance, ignoring the induced estrus, with AI 12 h after detection of the following spontaneous estrus. 2.3. Semen processing Semen was collected with an artificial vagina and estrous teaser ewes. All rams delivered ejaculates with acceptable volume (0.75–2 mL), concentration (⬎ 3.0 ⫻ 109 sperm/mL), total motility (⬎ 70%), and sperm morphology (⬍ 10% total sperm abnormalities). During the experiment, two consecutive ejaculates from each ram were collected within 10 min, pooled, and treated as a single ejaculate. Ejaculates from each ram were placed in a portable water bath at 33 °C and subjectively assessed (motility, volume and color). Sperm concentration was assessed with a photometer (Spermacue®, Minitub, Landshut, Germany). Fresh undiluted AI doses (200 ⫻ 106 sperm/ ewe; Experiments 1 and 3) or extended in UHT skim milk with antibiotics (final concentration 1000 ⫻ 106 sperm/mL, insemination dose 0.2 mL/ewe; Experiment 2) were maintained at 30 °C and used within 30 min after collection. 2.4. Estrus detection and AI The onset of estrus was determined twice daily during the first 72 h after the second PGF2␣ treatment in ewes in Experiment 1 (Synchrovine® and Synchrovine®-LD), and Experiment 3 (Synchrovine®-
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DE), or 17 d after PGF2␣ dose in Experiment 3 (Control, spontaneous estrus). Five vasectomized rams/100 ewes (Experiment 1) or 10 androgenized wethers/100 ewes (Experiment 3) were used as painted teaser animals. Wethers were treated with 100 mg testosterone cyclopentylpropionate im (Testosterona Ultra Fuerte®, Laboratorios Dispert, Montevideo, Uruguay), given on three occasions (14, 7 and 1 d, respectively, before the beginning of the estrus detection period). Paint was applied twice daily. Cervical AI was done using a speculum equipped with a light source and an insemination gun (Walmur Veterinary Instruments, Montevideo, Uruguay), slowly depositing the semen as deep as possible into the cervix. 2.5. Reproductive performance Estrous behaviour (ewes in estrus/treated) and its distribution within 72 h after the second PGF2␣ treatment was assessed (Experiments 1 and 3). Rates of conception (pregnant/inseminated ewes), prolificacy (fetuses/pregnant ewe), and fecundity (fetuses/inseminated ewes), were evaluated 30 d (Experiments 1 and 3) or 40 d (Experiment 2) after TAI by transrectal or transabdominal ultrasonography (Aloka® 500, Tokyo, Japan; 5.0 MHz linear-array or 3.5 MHz convex-array, respectively). 2.6. Statistical analyses Differences in estrous behaviour and its distribution, and rates of conception, prolificacy and fecundity among synchronization protocols or spontaneous estrus after AI were analyzed by ANOVA for categorical variables (PROC CATMOD; SAS). Data are presented as means, with P ⬍ 0.05 considered significantly different. 3. Results 3.1. Experiment 1 The results are shown in Fig. 1 and Table 1. The PGF2␣ dose did not significantly affect the proportion of ewes coming into estrus up to 72 h after the second PGF2␣ (95 vs 92%; P ⫽ NS), with a high concentration between 25 and 48 h (86 vs 73%, P ⫽ 0.07) for Synchrovine® and Synchrovine®-LD respectively. However, reproductive performance was better (P ⬍ 0.05) in Synchrovine® treated ewes compared to Synchrovine®-LD.
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Fig. 1. Distribution of estrous ewes treated with two doses ( □ Synchrovine®-LD, 80 g; Synchrovine®, 160 g) of PGF2␣ (delprostenate) given 7 d apart (n⫽127; Experiment 1). There were no significant differences between experimental groups.
3.2. Experiment 2 Reproductive performance (conception, prolificacy, and fecundity) for each group is shown (Table 2). There was no AI-time effect (42 vs 48 h, P ⫽ NS) within the PGF2␣ treatment interval (7 or 8 d apart). Nevertheless, there was a PGF2␣ treatment interval effect, with better reproductive performance (P ⬍ 0.05) in ewes given PGF2␣ 7 d apart compared to those treated 8 d apart. The interaction PGF2␣ treatment interval*AI time was significant (P ⬍ 0.05) just for prolificacy, but not for conception or final fecundity (P ⫽ NS). 3.3. Experiment 3 The proportion of ewes showing estrus between 25 and 48 h and the total number showing estrus by 72 h after the second PGF2␣ in Synchrovine®:DE group were 81 and 92%, respectively. Neither estrus detection
Table 1 Reproductive responses obtained with two doses of PGF2␣ (delprostenate) and cervical insemination with fresh semen in sheep (Experiment 1). Conception Synchrovine® (n ⫽ 64) Synchrovine®-LD (n ⫽ 63)
Prolificacy
Fecundity
0.42 (27/64)
1.11 (30/27)
0.47a (30/64)
0.24b (15/63)
1.13 (17/15)
0.27b (17/63)
a
Synchrovine®, two PGF2␣ treatments 7 d apart (160 g delprostenate) and TAI 42 h after second PGF2␣; Synchrovine®-LD, two PGF2␣ treatments 7 d apart (80 g delprostenate) and TAI 42 h after second PGF2␣; Conception, pregnant at 30 d after AI/inseminated ewes; Prolificacy, fetuses/pregnant ewe; Fecundity, fetuses/inseminated ewe. a,b Within a column, rates without a common superscript differed (P ⬍ 0.05).
(Synchrovine®-DE) nor GnRH at TAI (Synchrovine®GnRH) significantly improved (P⫽NS) the reproductive performance of synchronized ewes. All groups subjected to these synchronization protocols had lower (P ⬍ 0.05) fecundity than the Control group (spontaneous estrus, Table 3). 4. Discussion In the present study, both the amount of PGF2␣ and the interval between PGF2␣ treatments significantly affected reproductive performance of synchronized ewes. However, neither estrus detection (to determine time of AI) nor administration of GnRH at TAI significantly affected the reproductive outcome of the Synchrovine® protocol. The cumulative proportion of ewes showing estrus by 72 h after the second PGF2␣ treatment was similar between the full and half dose of PGF2␣ analog (Experiment 1), but the ewes in estrus between 25 and 48 h in the group treated with the full dose (Synchrovine®) tended to be higher, with a sharp onset of estrus. This could account for the better reproductive performance in this group. These results contradicted other basic experiments using a reduced dose of cloprostenol [16,17], but agreed with other studies [20] regarding estrus response following dinoprost tromethamine treatment (10 vs 5 mg) of young CLs (3.5 to 5 d old). The concept that a reduced dose of PGF2␣ would improve the reproductive performance, based on reduced side effects, was not supported. Perhaps some of the young CLs failed to respond to PGF2␣ [25], as observed in the Synchrovine® protocol, thereby reducing the number of ewes that were close to the time of ovulation at TAI.
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Table 2 Reproductive performance in sheep obtained with two PGF2␣ (delprostenate) at two intervals and cervical insemination (fresh semen) at two times (Experiment 2). Conception Synchrovine® (n ⫽ 145) Synchrovine®-48 (n ⫽ 145) Synchrovine®8-42 (n ⫽ 147) Synchrovine®8-48 (n ⫽ 146)
a
0.45 (65/145) 0.51a (74/145) 0.33b (48/147) 0.29b (42/146)
Prolificacy a
1.09 (71/65) 1.04a (77/74) 1.02a (49/48) 1.19b (50/42)
Fecundity 0.49a (71/145) 0.53a (77/145) 0.33b (49/147) 0.34b (50/146)
Synchrovine®, two PGF2␣ treatments 7 d apart and TAI 42 h after second PGF2␣; Synchrovine®-48, two PGF2␣ treatments 7 d apart and TAI 48 h after second PGF2␣; Synchrovine®8-42, two PGF2␣ treatments 8 d apart and TAI 42 h after second PGF2␣; Synchrovine®8-48, two PGF2␣ treatments 8 d apart and TAI 48 h after second PGF2␣; Conception, pregnant at 40 d after AI/inseminated ewes; Prolificacy: fetuses/pregnant ewe; Fecundity, fetuses/inseminated ewe. a,b Within a column, rates without a common superscript differed (P ⬍ 0.05).
Variations in the time of insemination after the last PGF2␣ had no significant effects (Experiment 2). As observed previously with this TAI protocol using fresh semen, pregnancy rates were similar after cervical inseminations both at 42 and 48 h [14,26], offering a reasonable time-window for use of this protocol in the field. However, reproductive performance was significantly affected by altering the interval between doses of PGF2␣ in the Synchrovine® protocol, with better results for treatments given 7 vs 8 d apart. Although estrus response and ovulation was not determined in this experiment, as in a previous study by Rubianes et al [12], we speculate that the interval between treatment and ovulation was more varied in ewes treated 8 d apart. This was probably due to a higher proportion of ovulations coming from the second follicular wave, resulting in TAI occurring too early for optimal fertility. Experiment 3 tested the effect of estrus detection, with only ewes in estrus inseminated, and GnRH at TAI. Firstly, AI of ewes showing estrus after PGF2␣ treatment did not improve the conception rate of this new synchronization protocol using fresh semen. The higher cumulative proportion of ewes that showed es-
trus between 25 and 48 h after the second PGF2␣ in the Synchrovine®-DE group, and the prolonged lifespan of fresh semen, may explain the similar conception rate achieved in the Synchrovine® TAI group. Therefore, we concluded that estrus detection is not required when using this protocol with fresh semen, reinforcing the advantage of TAI procedures. Secondly, giving a GnRH analogue concurrent with TAI did not improve the final reproductive outcomes of Synchrovine® protocol. Furthermore, the conception rate of this treatment tended to be lower (P ⫽ 0.09), but the prolificacy was higher (P ⫽ 0.07) than in the Synchrovine® group. These results agreed with previous reports where, despite the synchrony in ovulation achieved, giving GnRH within 24 to 44 h after a P4-eCG treatment failed to improve ewe fertility [27–29]. Other authors reported a tendency to increase litter size as a result of GnRH treatment immediately after the AI of ewes synchronized with a P4-eCG-PGF2␣ [30], or 30 h after a P4-PGF2␣ or P4-PGF2␣-eCG treatment [8]; this was attributed to GnRH increasing ovulation rates [30 –32]. In another study, the male effect was used after a short-interval, cloprostenol-based protocol to induce an earlier preovulatory LH surge and ovulation, thus im-
Table 3 Reproductive response obtained with variations of Synchrovine® protocol using cervical insemination with fresh semen in sheep (Experiment 3). Conception Synchrovine® (n ⫽ 111) Synchrovine®-DE (n ⫽ 102) Synchrovine®-GnRH (n ⫽ 104) Spontaneous Estrus (Control, n ⫽ 104)
ab
0.50 (55/111) 0.47ab (44/94) 0.38a (40/104) 0.59b (51/86)
Prolificacy a
1.13 (62/55) 1.18a (52/44) 1.28ab(51/40) 1.39b (71/51)
Fecundity 0.56a (62/111) 0.55a (52/94) 0.49a (51/104) 0.83b (71/86)
Synchrovine®, two PGF2␣ treatments 7 d apart and TAI 42 h after second PGF2␣; Synchrovine®-DE, two PGF2␣ treatments 7 d apart and AI 12 h after detected estrus; Synchrovine®-GnRH, two PGF2␣ treatments 7 d apart plus 8 g GnRH (busereline acetate) at TAI (42 h after second PGF2␣); Spontaneous Estrus, pre synchronized ewes with one dose of PGF2␣ 17 d in advance, ignoring the induced estrus, and AI 12 h after detected spontaneous estrus; Conception, pregnant 30 d after AI/inseminated ewes; Prolificacy, fetuses/pregnant ewe; Fecundity, fetuses/ inseminated ewe. a,b Within a column, rates without a common superscript differed (P ⬍ 0.05).
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proving fertility compared to a progesterone-based protocol [13]. Consequently, a GnRH dose was expected to favor the Synchrovine® TAI protocol. Assuming that ovulation in Synchrovine® protocol occurs approximately 60 h after the second PGF2␣ treatment [12,13], and that the physiological LH-surge is ⬃24 h before ovulation [33], the LH surge should be ⬃36 h after the last PGF2␣. If the GnRH was given at TAI (42 h), perhaps this promoted a second LH surge 2– 4 h later [21,28]. In this regard, the GnRH was probably too late to augment the endogenous LH surge to target a timed ovulation. Although it is logistically easier to give GnRH concurrent with TAI, earlier administration should be considered in future trials. Finally, the Synchrovine® protocol and the studied alternatives had lower reproductive outcomes compared to spontaneous estrus and AI based on estrus detection. An environment dominated by lower progesterone concentration during the growth phase of the ovulatory follicle, stimulating a faster growth rate, and a larger follicular size, appeared to be a cause of the lower ovulation rate, conception, and prolificacy achieved with Synchrovine® protocol compared to ewes AI in spontaneous estrus [15]. In conclusion, reproductive performance of the Synchrovine® protocol was: a) better when using 160 versus 80 g delprostenate; b) better when using a 7 d interval compared to an 8 d interval between PGF2␣ treatments; c) similar with insemination at prefixed time or AI 12 h after estrus detection; and d) not improved by giving GnRH at TAI.
Acknowledgments This work was supported by funds from Universidad de la República (CSIC 600/6015) and MGAP-DILAVE “M.C. Rubino”. We acknowledge “Coraje” Farm (Dighiero-Triay family), “Piedra Mora” Farm (FilliolBarreiro family) and EEMAC (Facultad de Agronomía, UdelaR, Paysandú, Uruguay) for allowing us to use their facilities and animals. Thanks also to E. Campos and S. Kmaid from Universal Lab and J. Amaro from Dispert for donating delprostenate and testosterone, respectively. We are also grateful to J.L. Alabart for statistical analysis; to G. McCann for revising the manuscript; and to S. Forichi, M. Correa, A. Araujo, E. Filliol, and G. Stoletniy for their support during the trials. The authors do not have any financial or personal relationships with other people or organizations that could have inappropriately biased or influenced this work.
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