Cloprostenol enhances sexual behaviour and semen quality in growing lambs more effectively than Dinoprost
Abstract
Analogues of PGF2α have been used in boars with positive results on sexual behaviour, but limited impact on semen quality. The objective of this study was to determine whether the administration of Dinoprost tromethamine or D-Cloprostenol enhances the sexual behaviour and semen quality of sexually inexperienced growing lambs during the onset of the breeding season. The study lasted 10 weeks, beginning during the non-breeding season, with 24 sexually inexperienced Katahdin lambs (5.1 ± 0.5 months old). Three groups of eight lambs each received: (1) vehicle treatment (group CON); (2) 10 mg of Dinoprost tromethamine (group DIN); or (3) 0.15 mg of Cloprostenol dextrogy (group CLOP). The treatments were all applied im 5 min before the tests of sexual behaviour, or 20 min before the collection of semen. Sexual behaviour was evaluated twice weekly in a pen test with a non-oestrous ewe, and semen was collected once weekly with an artificial vagina. The latency to begin courtship was significantly shorter in both groups that received analogues of PGF2α than in CON lambs. The number of ano-genital sniffs, flehmens and lateral approaches was greater in both treated groups than in CON lambs (p < .05). Lambs from DIN group mounted significantly more than CLOP and CON lambs, which did not differ. However, CLOP lambs showed significantly greater mating efficiency (i.e. ejaculations/total mounts) than CON lambs, which exhibited significantly greater mating efficiency that DIN lambs. Lambs treated with CLOP produced significantly more semen volume with more mass motility than those treated with DIN and CON (without differences between DIN and CON). Overall, we concluded that the administration of cloprostenol or dinoprost before sexual evaluation in young rams enhances their sexual behaviour. Cloprostenol had greater effects than dinoprost. The administration of cloprostenol before semen collection also triggered the ejaculation of a greater volume of semen, with greater mass motility. These results open interesting possibilities to study deeply the use of simple and cost-effective treatments to improve the reproductive results of young growing rams.
1 INTRODUCTION
Ram reproductive performance is highly variable among breeds and is affected by the environment (Sánchez-Dávila et al., 2020), age (Santos et al., 2015) or their sexual experience (Aké-Villanueva et al., 2019). Hormone treatments can increase the reproductive performance of rams (Ungerfeld et al., 2014, 2020), since testosterone propionate (Tejada et al., 2017) or eCG (Ungerfeld et al., 2014) has been administered to increase the ram ability to stimulate the number of ewes that respond to the ram effect. Administration of eCG also enhances semen quality in goats during the non-breeding season, although it may induce the synthesis of anti-eCG antibodies (Beracochea et al., 2018), with the associated risk of decreasing its effectiveness after repeated administrations.
Analogues of PGF2α have been used in boars with positive results on sexual behaviour, but limited impact on semen quality (Estienne, 2014). Dinoprost tromethamine and D-Cloprostenol are the two analogues most frequently used in boars, but doses of 10 mg of Dinoprost tromethamine—that are effective in boars—did not have positive effects on sexual behaviour of male rams (Ungerfeld et al., 2020). However, the administration of cloprostenol twice weekly improved some characteristics of the ejaculate in rams (Olfati et al., 2013). Therefore, the response is probably related to the analogue, dose and frequency of administration, opening interesting possibilities to develop protocols for rams. Moreover, while the use of some hormones is socially debated, prostaglandin analogues are used without problem throughout the world, reinforcing the potential application of treatments based on these hormones.
Therefore, the objective of this study was to determine whether the administration of Dinoprost tromethamine or D-Cloprostenol enhances the sexual behaviour and semen quality of sexually inexperienced growing lambs during the onset of the breeding season.
2 MATERIALS AND METHODS
2.1 Location of the experiment and animals used
The study was carried out at the Laboratorio de Reproducción Animal of the Facultad de Agronomia, Universidad Autonoma de Nuevo León (UANL), located at 375 m above sea level and 25°53′N, 100°02′W. The experiment was approved by the Ethics and Animal Welfare Committee of the Facultad de Medicina Veterinaria y Zootecnia of the UANL (14/2020).
The study lasted ten weeks during the non-breeding season (spring/summer). Twenty-four sexually inexperienced young Katahdin lambs (5.1 ± 0.5 months old and 33.5 ± 6.0 kg, mean ± SD) were used. The lambs were housed in individual pens (2 m × 1 m) and offered a concentrate with 16% CP (Ovejero 1NP, Forajera Elizondo, Apodaca, Mexico) and free access to water. Before starting the experiment, the lambs were orally dewormed with albendazole (5 mg/kg) and closantel (10 mg/kg) (Microtel, Microsules, Uruguay). They also received 200,000 IU of vitamin A, 30,000 IU of vitamin D, 20 IU of vitamin E im (ADE Prode, Internacional Prode, Mexico) and the 10-way vaccine (Bovinmune 10, Lapisa, Mexico) sc. The scrotal circumference of the lambs was measured weekly with a tape, holding and descending the testicles, referencing the middle part of the scrotum, before collecting semen (see Section 2.4).
2.2 Experimental treatments
Three groups of eight lambs each were blocked according to their body weight and allocated to the following treatments: (1) 2 ml of saline solution (group CON); (2) 10 mg of Dinoprost tromethamine (Lutalyse, Zoetis, Jalisco, Mexico; group DIN); or (3) 0.15 mg of Cloprostenol dextrogy (Prostagenol-D, PRODE Internacional, Jalisco, Mexico; group CLOP). The treatments were all applied im 5 min before the tests of sexual behaviour, or 20 min before the collection of semen.
2.3 Sexual behaviour
This test was held twice weekly, on Tuesdays and Wednesdays, totalling 20 tests. An ewe was placed in a separate pen that gave access only to the ram lamb being tested so as to prevent stimulation from other lambs. For this test, a non-oestrous ewe (treated with a vaginal sponge with 60 mg of medroxyprogesterone inserted 3 days before the test) remained restrained to facilitate the sexual unfolding of each lamb. The test was performed in a 2 m × 1 m pen, for 5 min. The following measures of sexual behaviour were recorded: latency to display of courtship, ano-genital sniffs, lateral approaches, flehmen reflexes, mount attempts, mounts and ejaculations. Mating efficiency was calculated as the ratio of ejaculations to total mounts (mounts+ejaculations).
2.4 Seminal quality
Semen was collected from each ram lamb once weekly, every Friday, totalling 10 collections. For this, a 15 cm × 5 cm artificial vagina loaded with 39°C pre-heated water was used. Semen was collected into 15 ml Falcon tubes. For this test, an oestrous ewe (treated with 1 mg of estradiol benzoate; Internacional Prode) was used, remaining restrained during the procedure. Each lamb was given a maximum of 5 min to ejaculate in the artificial vagina. The volume obtained was recorded from the graduated collection tubes. The samples obtained were placed at 37°C. To evaluate the mass motility, a drop of semen was placed on a slide pre-heated to 37.5°C and was evaluated on a scale of 0 to 5 with the aid of a microscope (Primo Star Carl Zeiss). The percentage of sperm with progressive motility (0% to 100%) was assessed placing a coverslip and observing at the 10 ˟ objective. The sperm concentration was determined without dilution with a photometer (SDM1, Minitube). The total number of spermatozoa ejaculated and the total number of spermatozoa with progressive motility ejaculated were calculated.
2.5 Statistical analysis
Data were analysed with mixed models, including the treatment (CON, DIN or CLOP), the number of sexual test or semen collection, as main effects, and their interaction. Time was included in the model to control its effect adjusting the LSmeans and to determine the interaction with treatments, but not due to a direct interest of changes with time unrelated to the treatments, as it is expected that most variables increase due to be growing, developing lambs. The compound symmetry covariance structure was used, and time was considered a repeated measure. Post hoc differences were based on the pdiff analysis of SAS software. Differences were considered as significant when p ≤ .05, and as tendencies when .05 < p ≤ .1.
3 RESULTS
The scrotal circumference varied with time (p < .0001), with no interaction with treatment. The scrotal circumference of CLOP lambs was greater than that of DIN lambs (29.3 cm vs. 27.1 cm; pooled SEM = 0.48; p = .002), and CON lambs also tended to have greater circumference (28.3 cm; p = .08) than DIN lambs.
The effects of the main factors on the lambs' sexual behaviour are presented in Table 1. Except for mount attempts, ejaculations and time to the first ejaculation, there were significant effects of treatments on all the behaviours studied. The latency to courtship was shorter in both groups that received analogues of PGF2α than in CON lambs (p < .005). The number of ano-genital sniffs, flehmens and lateral approaches was greater in both treated groups than in CON lambs (p < .05; Table 1). Lambs from DIN group mounted significantly more than CLOP and CON lambs (p < .0001 for mounts and mounts+ejaculations), which did not differ (Table 1). However, CLOP lambs showed significantly greater mating efficiency (i.e. ejaculations/total mounts) than CON lambs (p = .0002), which exhibited significantly greater mating efficiency that DIN lambs (p = .04; Table 1). There were changes along time in all the inspective behaviours, mount attempts and the time to first mating (Table 1), but no interaction between treatments and time.
| CON | DIN | CLOP | Pooled SEM | Treatment | Time | Treatment × Time | |
|---|---|---|---|---|---|---|---|
| Inspective behaviours | |||||||
| Lapse to courtship onset (s) | 4.6a | 2.4b | 2.7b | 0.5 | 0.005 | 0.004 | ns |
| Ano-genital sniffings | 3.6b | 4.7a | 4.3a | 0.2 | 0.0009 | <0.0001 | ns |
| Flehmen | 0.4b | 0.7a | 0.6a | 0.1 | 0.03 | 0.0009 | ns |
| Precopulatory behaviours | |||||||
| Lateral approaches | 1.9b | 3.9a | 3.1a | 0.3 | <0.0001 | ns | ns |
| Mount attempts | 0.6 | 0.8 | 0.7 | 0.1 | ns | 0.03 | ns |
| Mounts and ejaculations | |||||||
| Mounts | 2.9b | 4.5a | 2.8b | 0.3 | <0.0001 | ns | ns |
| Ejaculations | 1.2 | 1.2 | 1.2 | 0.1 | ns | ns | ns |
| Time to first ejaculate (s) | 46.4 | 56.3 | 52.0 | 4.6 | ns | 0.009 | ns |
| Total mounts | 4.1b | 5.8a | 3.9b | 0.3 | <0.0001 | ns | ns |
| Ejaculations/Total mounts | 0.35b | 0.28c | 0.48a | 0.02 | <0.0001 | ns | ns |
Main effects and LSmean values for each treatment of semen variables are presented in Table 2. Treatment affected semen volume and mass motility (p = .005 and p = .046 respectively) and tended to modify its concentration (p = .08). Lambs treated with CLOP produced more volume (p = .005) with greater mass motility (p = .046) than those treated with DIN and CON, (without differences between DIN and CON).
| CON | DIN | CLOP | Pooled SEM | Treatment | Time | Treatment × Time | |
|---|---|---|---|---|---|---|---|
| Semen volume (ml) | 0.28b | 0.33b | 0.43a | 0.03 | 0.005 | 0.0073 | ns |
| Mass motility | 1.8b | 1.8b | 2.3a | 0.2 | 0.046 | 0.0013 | ns |
| Progressive motility sperm (%) | 35.9 | 38.8 | 44.5 | 5.1 | ns | ns | ns |
| Sperm concentration (×106) | 2303 | 2126 | 2693 | 183 | 0.08 | <0.0001 | ns |
| Total sperm ejaculated (×106) | 1007 | 1065 | 1350 | 131 | ns | 0.0006 | ns |
| Total progressive motile sperm ejaculated (×106) | 646 | 670 | 861 | 103 | ns | 0.05 | ns |
4 DISCUSSION
In accordance with the hypothesis of the study, the application of prostaglandin analogues had a positive effect on sexual behaviour and seminal quality during the reproductive development period of male lambs. Although there was a direct effect, without a cumulative effect over time (no interactions between treatments and time), the single administration triggered changes in both responses. In a recent study, we did not observe any positive effect of the administration of dinoprost on rams sexual behaviour (Ungerfeld et al., 2020), so it remains to be determined, which are the conditions that allowed us to detect the positive impact in this study. The main difference between the previous study and this one is that in the preceding study, the treatments were applied to adult rams. On the contrary, in the present experiment treatments were applied during sexual development of male lambs, when animals are probably more likely to express differences in the responses to stimulation of their physiological capacities. Therefore, before discarding its possible use, it would be interesting to determine whether sexual behaviours in adult rams can be impacted by greater doses, prolonged treatments or other analogues of prostagladins.
Treated males displayed courtship behaviour earlier and also displayed more inspective and pre-copulatory behaviours (ano-genital sniffings, flehmen and lateral approaches); therefore, opening interesting possibilities to study whether similar treatments can be used effectively to enhance rams' ability as teasers and to stimulate ovulation in non-cyclic ewes (see reviews: Delgadillo et al., 2009; Ungerfeld et al., 2004). In this context, rams sexual behaviour is a determinant of the females response intensity (Perkins & Fitzgerald, 1994), so it remains to be determined whether similar treatments are also effective in adult rams, during the non-breeding season. In this sense, hormonal treatments such as the administration of eCG increased the effectiveness of rams of mid-intensive seasonal breeds to stimulate the ovulation of anoestrous ewes (Ungerfeld et al., 2014). However, a possible limitation of treatments with analogues of PGF2α is the short half-life of the hormone (Homeida & Cooke, 1983), limiting its possible application only to conditions of intensive management.
To the best of our knowledge, the mechanisms and pathways by which the prostaglandin or its analogues enhance sexual behaviour have not been elucidated even in other more studied species. It has been speculated that in boars, analogues of PGF2 might induce an increase in testosterone secretion (Estienne & Harper, 2000), but the positive effects are observed also in castrated pigs (Zamora et al., 2010), so it appears as more probably a direct action at the central nervous system (Estienne, 2014). In this sense, Burne et al. (2002) reported that dinoprost promotes the activity of neurons in several hypothalamic nuclei in pigs. In rats, there are some pioneer studies using PGE2, but it should be considered that the mechanisms of action might differ from those of PGF2 and its analogues. Blumberg (1991) provoked increases in hypothalamic temperature associated with a reduction in the post-ejaculatory refractoriness after administering PGE2 directly into the cerebral aqueduct. Moreover, castrated rats treated with testosterone increased more their number of copulas when PGE2 was intracranially administrated (Clemens & Glaude, 1977). Although the mechanisms might differ in different species, the scarce information available allows to speculate that these treatments probably have at least a central action promoting lambs' sexual behaviour. It would be interesting to determine whether similar mechanisms are also active in ruminants and whether these treatments could be useful to induce permanent changes in rams' sexual behaviour.
In general, the administration of cloprostenol increased semen volume and mass motility, whereas dinoprost had no effects on these variables. The mechanisms by which the administration of analogues of PGF2α might enhance semen collection have not been elucidated, but in one hand, Estienne (2014) proposed that analogues of PGF2α stimulate sexual motivation in boars and thus probably stimulated the ejaculatory process, based on its action in one or more areas of the central nervous system. A non-opposed explanation is that PGF2α stimulates ejaculation as it triggers the contractility of the muscles of the genital tract involved in the process (Cosentino et al., 1984). In fact, even when ejaculation is stimulated with electroejaculation, the administration of analogues of PGF2α concurrently with oxytocin shortens the time required for semen collection with electroejaculation in bucks (Ungerfeld et al., 2018). It would be interesting to determine whether the effect of this treatment persists and thus allow collection of more doses of semen and/or increases the numbers of females that ram is capable of impregnating.
5 CONCLUSIONS
Overall, we concluded that the administration of cloprostenol or dinoprost before sexual evaluation in young rams enhances their sexual behaviour. Cloprostenol had greater effects than dinoprost. The administration of cloprostenol before semen collection also triggered the ejaculation of a greater volume of semen, with greater mass motility. These results open interesting possibilities to study deeply the use of simple and cost-effective treatments to improve the reproductive results of young growing rams.
ACKNOWLEDGEMENTS
The authors acknowledge the Joint Postgraduate Program of the UANL and for the financial support received for this study, which is part of the Master's Program in Animal Science. Ing. Víctor Adrián Hernández Melo received a scholarship from CONACYT (Mexico).
CONFLICT OF INTEREST
The authors declare no conflict of interest.
AUTHORS CONTRIBUTIONS
R. Ungerfeld involved in conceptualization, writing, validation, review and formal analysis. F. Sánchez-Dávila involved writing, review, investigation and methodology. V.A. Gernandez-Melo involved writing, investigation and methodology. R. A. Ledezma Torres, H. Bernal-Barrágan and C. Luna-Palomares involved writing and review.
Open Research
DATA AVAILABILITY
The data that support the findings of this study are available from the corresponding author upon reasonable request.
