A. Casao

Melatonin prevents capacitation and apoptotic-like changes of ram spermatozoa and increases fertility rate.

Abstract:  We recently demonstrated the presence of melatonin in ram seminal plasma and differences in its concentration in this fluid between the breeding and nonbreeding season. In this study, we investigate the hypothesis that in vitro treatment with melatonin affects ram sperm quality, and that this is reflected in the in vitro fertilization (IVF) results. Semen from nine rams was collected during the nonreproductive season and treated with 1 μm, 10 nm and 100 pm melatonin. Samples were incubated at 39°C and 5% CO2, and motility, viability, capacitation status and phosphatidylserine (PS) translocation were assessed before and after melatonin addition, either 1 or 3 hr of incubation. Fertility rate of the melatonin‐treated samples was determined by means of IVF. Although melatonin failed to affect both sperm kinematic parameters and viability, the exposure of ram spermatozoa to melatonin has a direct effect, decreasing capacitation and PS translocation at 1 μm, and increasing short‐term capacitation at 100 pm, which caused an increased oocyte fertilization rate following IVF. Furthermore, cleavage rate of oocytes fertilized with 100 pm melatonin‐treated spermatozoa was higher than that with 1 μm melatonin and control samples (P < 0.1). These results prove that melatonin has a direct effect on ram spermatozoa in the nonreproductive season, which can be explained, at least in part, by the melatonin capacity as a reactive oxygen species scavenger and antioxidant. These findings might help to select the optimal experimental conditions for IVF and to improve sperm preservation protocols.

Effects of melatonin implants during non-breeding season on sperm motility and reproductive parameters in Rasa Aragonesa rams.

The effect of melatonin implants administered during non-breeding season in Rasa Aragonesa rams on sperm motility parameters and other reproductive traits was assessed. In a first experiment, two Rasa Aragonesa rams were implanted (with melatonin group M), remaining other two males as control group (C). Semen of each group was collected from 1 May to 23 June, twice or three times a week, and motility parameters were assessed using a computer-assisted sperm analysis system. Melatonin increased the percentage of progressive motile spermatozoa, particularly during 46-75 days after melatonin implantation (p < 0.01). In experiment 2, M and C in vitro fertilization ability had been determined by zona-pellucida binding assays, using spermatozoa from experiment 1, obtained 60-70 days after melatonin was implanted. A significantly higher number of spermatozoa attached per oocyte was observed in frozen-thawed immature ovine oocytes incubated with sperm from M animals than in those incubated with sperm from the C group (p < 0.01). Finally, a field assay (experiment 3) was performed. In this case, five Rasa Aragonesa rams were implanted with melatonin and three remained as control group. Sperm doses from those animals were used for artificial insemination of 2608 Rasa Aragonesa ewes from 39 different farms at non-breeding season. Fertility, litter size and fecundity were studied. Semen from melatonin implanted rams seemed to increase both fertility and fecundity in ewes inseminated with spermatozoa obtained 46-60 days after implantation (p < 0.1). Thus, melatonin treatment in rams during non-breeding season modifies sperm motility parameters and seems to improve the fertilization parameters obtained.

Seasonal variations of melatonin in ram seminal plasma are correlated to those of testosterone and antioxidant enzymes

BackgroundSome breeds of sheep are highly seasonal in terms of reproductive capability, and these changes are regulated by photoperiod and melatonin secretion. These changes affect the reproductive performance of rams, impairing semen quality and modifying hormonal profiles. Also, the antioxidant defence systems seem to be modulated by melatonin secretion, and shows seasonal variations. The aim of this study was to investigate the presence of melatonin and testosterone in ram seminal plasma and their variations between the breeding and non-breeding seasons. In addition, we analyzed the possible correlations between these hormones and the antioxidant enzyme defence system activity.MethodsSeminal plasma from nine Rasa Aragonesa rams were collected for one year, and their levels of melatonin, testosterone, superoxide dismutase (SOD), glutathione reductase (GRD), glutathione peroxidase (GPX) and catalase (CAT) were measured.ResultsAll samples presented measurable quantities of hormones and antioxidant enzymes. Both hormones showed monthly variations, with a decrease after the winter solstice and a rise after the summer solstice that reached the maximum levels in October-November, and a marked seasonal variation (P < 0.01) with higher levels in the breeding season. The yearly pattern of GRD and catalase was close to that of melatonin, and GRD showed a significant seasonal variation (P < 0.01) with a higher activity during the breeding season. Linear regression analysis between the studied hormones and antioxidant enzymes showed a significant correlation between melatonin and testosterone, GRD, SOD and catalase.ConclusionsThese results show the presence of melatonin and testosterone in ram seminal plasma, and that both hormones have seasonal variations, and support the idea that seasonal variations of fertility in the ram involve interplay between melatonin and the antioxidant defence system.

Undernutrition and exogenous melatonin can affect the in vitro developmental competence of ovine oocytes on a seasonal basis.

This study evaluated the effects of exogenous melatonin and level of nutrition on oocyte competence, in vitro fertilization (IVF), and early embryonic development in sheep during seasonal anoestrus (SA) and the reproductive season (RS). Adult Rasa Aragonesa ewes were assigned randomly to one of four treatment groups in two experiments based on a 2 x 2 x 2 factorial design. Individuals were treated (+MEL) or not treated (-MEL) with a subcutaneous implant of melatonin for 42 days and then were fed 1.5 (Control, C) or 0.5 (Low, L) times the daily maintenance requirements for 20 days. Ewes were synchronized and mated at oestrus (Day = 0). On Day 5, ovaries were collected and oocytes were used for IVF. Season had a significant (p < 0.01) effect on the number of oocytes recovered (RS: 19.6 +/- 1.0; SA: 14.5 +/- 1.0) and the number of healthy oocytes (RS: 13.9 +/- 0.7; SA: 9.0 +/- 0.7). In the RS, neither nutrition nor melatonin had a significant effect on the evaluated oocytes quality parameters although melatonin implants appeared to reduce the number of unhealthy oocytes in the undernourished group (p < 0.05). During SA, in undernourished ewes exogenous melatonin tended to increase the number of healthy (L+MEL: 9.4 +/- 1.0, L-MEL: 7.6 +/- 1.4; p < 0.1), and significantly improved both cleaved oocytes (L+MEL: 7.0 +/- 0.7, L-MEL: 4.1 +/- 0.9; p < 0.05) and blastocyst rate (L+MEL: 37.2, L-MEL: 21.9%; p < 0.05). In conclusion, oocyte competence in ewes was affected by season, and melatonin implants appeared to improve developmental competence in the seasonal anoestrous period, particularly in experimentally undernourished ewes.

New Insights into the Mechanisms of Ram Sperm Protection by Seminal Plasma Proteins

ABSTRACT To provide new insights into the mechanisms through which seminal plasma proteins (SPP) are able to protect spermatozoa, we tested the hypothesis that apoptosis can contribute to the negative effect of refrigeration on ram spermatozoa, and that SPP prevent this damage. Having proved the presence of key constituents of apoptosis-related pathways in ram sperm protein extracts, we carried out a comparative analysis of the effects of the addition of SPP before refrigeration (15°C, 30 min) and induced-apoptosis with betulinic acid or fibroblast-associated receptor ligand, assessing sperm quality parameters and apoptotic markers. The protective effect of SPP on plasma membrane integrity and potential, motility and mitochondrial inner membrane potential, and surface (cardiolipin content) was evidenced in refrigerated and induced-apoptosis samples. The addition of SPP resulted in lower values of phosphatidylserine externalization, DNA damage, and caspase activity. Therefore, apoptosis in fresh or refrigerated ram spermatozoa can occur due to activation of both the extrinsic and the intrinsic mediated pathway, and SPP might interfere with both pathways. The addition of SPP also resulted in higher proportions of viable, noncapacitated sperm and fertilizing ability (ZBA rate). This report demonstrates that SPP support survival of ram spermatozoa acting not only at the plasma membrane but also by inhibition of capacitation, and proposes the possibility that SPP might interfere with the extrinsic and the intrinsic apoptotic pathways. This opens new, interesting perspectives for the study of cellular regulatory mechanisms in spermatozoa that could be crucial for the improvement of ram semen preservation protocols.

Effects of exogenous melatonin treatment on out-of-season ram fertility

Abstract Three field experiments were performed to determine the effects of melatonin implants on the fertility of rams. The experiments were performed on three breeds (Rasa Aragonesa, Assaf and Manchega) in three commercial farms in Spain. Melatonin-treated rams (M) and non-implanted control rams (C) mated either ewes oestrous-induced with FGA intravaginal sponges (Rasa Aragonesa and Assaf farms) or melatonin-treated ewes during the anoestrous period (Manchega farm). Scrotal circumference (SC) was measured at melatonin implantation and at ram introduction (nearly 45 d later). Lambing rate, litter size, and fecundity were recorded at parturition. Melatonin implants in rams increased the number of lambs born per ewe in Rasa Aragonesa (19%), Assaf (9%), and Manchega (7%) because of the higher lambing rates exhibited by ewes mated with melatonin-treated rams (Rasa Aragonesa: 80.2% vs 70.5%, P<0.05; Assaf: 74.2% vs 68.0%, NS; Manchega: 90.0% vs 81.5%, P<0.05; for M and C groups, respectively). As a result, the number of extra lambs born per treated ewe was 0.17, 0.10, and 0.10 in the Rasa Aragonesa, Assaf, and Manchega ewes, respectively. In the M and C groups of Rasa Aragonesa rams, mean scrotal circumference increased significantly between the start of the experiments and the onset of mating, but the two groups did not differ significantly at the implantation or at the onset of mating. In the melatonin-treated and control Assaf rams, scrotal circumference increased over the course of the experiments but, at the onset of mating, the melatonin-treated rams had higher SC than did the control rams. Melatonin-treated Manchega rams had scrotal circumferences that were significantly greater than those of the control rams. In the Rasa Aragonesa and Manchega breeds, treating rams using melatonin implants significantly increased the net lamb production of ewes in field conditions. In the Assaf breed, the number of extra lambs born per ewe mated with melatonin treated rams was lower than in the other breeds, even though rams exhibited a significant increase in scrotal circumference. Further research involving the study of semen quality has been planned.

Repeated superovulation using a simplified FSH/eCG treatment for in vivo embryo production in sheep

This study investigated the efficacy of a simplified repeated superovulation treatment (eCG plus FSH in a single dose, rather than the usual protocol of six decreasing doses of FSH) in the in vivo embryo production in Ojalada donor ewes during the breeding season. In vitro viability after vitrification and warming of embryos recovered from both treatments was also assessed. In addition, the study examined the effects of the concentration of anti-eCG antibodies before each eCG/FSH treatment on in vivo embryo production. Thirty-eight females at the end of their reproductive lives were given the decreasing (n = 19) or simplified (n = 19) superovulatory treatment up to three times at intervals of ≥ 50 d. The onset of estrus was 5 h earlier (P < 0.05) among ewes that received the eCG/FSH protocol (25.2 ± 0.80 h) than it was among those that received the decreasing superovulatory treatment (30.1 ± 1.0 h), but the two treatments did not differ significantly in ovulation rates or the number and viability of embryos recovered. Both of the superovulatory protocols were significantly (P < 0.05 to P < 0.01) less effective after the first application. After three superovulatory treatments, the average number of viable embryos per ewe was 14.1 ± 2.3 and 13.7 ± 2.5 in the decreasing and simplified protocols, respectively. High anti-eCG antibody concentrations just before the superovulatory treatment with eCG/FSH were associated with a significant decrease (P < 0.05) in the rates of fertilization, viability, and freezability, especially in the second and third recoveries. Repeated superovulatory treatments with eCG/FSH can provide an efficient means of producing high quality embryos in the ewes of endangered breeds at the end of their reproductive lives, although further studies are needed to characterize the response associated with high concentrations of anti-eCG antibodies.

Evidence of melatonin synthesis in the ram reproductive tract.

Melatonin is a ubiquitous molecule found in a wide range of fluids, one of them being ram seminal plasma, in which it can reach higher concentrations than those found in blood, suggesting an extrapineal secretion by the reproductive tract. In order to identify the source of the melatonin found in ram seminal plasma, we first tried to determine whether the melatonin levels were maintained during the day. For this purpose, melatonin concentrations were measured in seminal plasma obtained from first ejaculates of six rams at 6:00 a.m. in total darkness, at 10:00 a.m. and at 14:00 p.m. The melatonin concentration was higher (p < 0.05) in ejaculates collected at 6:00 a.m. than at 10:00 and 14:00. There was no statistical difference between the latter. To further corroborate an extrapineal secretion of melatonin, the presence of the two key enzymes involved in melatonin synthesis, arylalkylamine‐N‐acetyltransferase (AANAT) and N‐acetylserotonin‐O‐methyltransferase (ASMT) was analyzed by RT‐PCR, q‐PCR and Western‐blot in ram testes, epididymis, and accessory glands. The RT‐PCR showed the presence of the m‐RNA codifying both AANAT and ASTM in all the tissues under study, but the q‐PCR and Western‐blot revealed that gene expression of these enzymes was significantly higher in the testis (p < 0.05). Immunohistochemistry confirmed the presence of AANAT and ASMT in the testis and revealed that they were found in the Leydig cells, spermatocytes, and spermatids. Also, measurable levels of melatonin were found in testicular tissue and the tail of the epididymis. In conclusion, our study indicates that the testes are one of the likely sources of the high levels of melatonin found in ram seminal plasma, at least during the day.

The effect of exogenous melatonin during the non-reproductive season on the seminal plasma hormonal profile and the antioxidant defence system of Rasa Aragonesa rams.

The aim of this study was to analyze the effect of melatonin implants, during the non-reproductive season, on the content of melatonin, testosterone and 17-β estradiol levels, and the antioxidant enzymes glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase of seminal plasma in Rasa Aragonesa rams. Five rams were implanted with melatonin, and four others were used as a control group. Seminal plasma was separated from ejaculates collected one week before melatonin treatment until 21 weeks after melatonin placement, and the hormonal levels and the antioxidant enzyme activity were determined. Exogenous melatonin treatment significantly (P<0.05) increased the levels of endogenous melatonin in seminal plasma immediately, and this effect lasted for 14 weeks. Testosterone and 17-β estradiol levels significantly (P<0.05) increased four and eight weeks after melatonin treatment, respectively. As regards the antioxidant enzymes, melatonin treatment significantly increased (P<0.05) glutathione peroxidase and glutathione reductase activity only, and had no effect on superoxide dismutase and catalase. Therefore, melatonin treatment during the non-breeding season modifies the seminal plasma hormonal profile and some antioxidant enzyme activity in Rasa Aragonesa rams.

Effects of melatonin and undernutrition on the viability of ovine embryos during anestrus and the breeding season

This study examined the effects of melatonin and level of nutrition on embryo yield during anestrous and breeding season. Adult Rasa Aragonesa ewes were assigned randomly to one of the four treatment groups in two experiments using a 2x2x2 factorial design. Individuals were treated (+MEL) or not treated (-MEL) with a subcutaneous implant of melatonin for 42d (Melovine, CEVA) and fed 1.5 (control, C) or 0.5 (low, L) times the daily maintenance requirements for 20d. Ewes were mated at oestrus (Day=0) and embryos were recovered on Day 5. Level of nutrition and melatonin supplements did not have a significant effect on ovulation rate or the number of recovered ova per ewe in the Reproductive Season (RS) and the Anestrous Season (AS). During the RS, undernutrition reduced the number of viable embryos per ewe (C: 1.1+/-0.2; L: 0.6+/-0.2; P<0.05); however, the number of viable embryos per ewe in the L+MEL group (0.2+/-0.15) was significantly lower than it was in the L, C+MEL and C groups (0.9+/-0.3, 1.2+/-0.3, 1.0+/-0.4, respectively; P<0.05). In the AS, nutrition did not have a significant effect on the number of viable embryos per ewe, although melatonin supplements might have improved rates slightly. Embryo viability rate (% viable embryos/embryos recovered) was unaffected by melatonin supplements or level of nutrition in the RS and the AS. Season had a strong effect on the number of viable embryos per functional corpus luteum among ewes in the L+MEL group, only (RS: 0.2+/-0.1; AS: 0.6+/-0.2; P<0.05). In conclusion, undernutrition impaired the viability of sheep embryos in the RS, particularly among ewes that were given melatonin supplements subcutaneously, but melatonin appeared to improve embryo quality in the AS, which suggests that the mechanisms involved in the interactive effects of melatonin and nutrition on embryo development are influenced by season.