Steroid hormone receptors and direct effects of steroid hormones on ram spermatozoa.

This study was based on the assumption that steroid hormones present in the female genital tract may have a rapid effect on ram spermatozoa by interaction with specific surface receptors. We demonstrate the presence of progesterone (PR) and estrogen (ER) receptors in ram spermatozoa, their localization changes during in vitro capacitation and the actions of progesterone (P4) and 17ß-estradiol (E2) on ram sperm functionality. Immunolocalization assays revealed the presence of PR mainly at the equatorial region of ram spermatozoa. Western blot analyses showed three bands in ram sperm protein extracts of 40-45 kDa, compatible with those reported for PR in the human sperm membrane, and both classical estrogen receptors (66 kDa, ERα and 55 kDa, ERß). ERα was located in the postacrosomal region of all the spermatozoa and ERß on the apical region of 63.7% of the cells. The presence of ERß was correlated with the percentage of non-capacitated spermatozoa evaluated by chlortetracycline staining (R = 0.848, P < 0.001). This significantly decreased after in vitro capacitation and nearly disappeared when acrosome reaction was induced. The addition of P4 and E2 before in vitro capacitation resulted in a higher (P < 0.001) acrosome-reacted sperm rate compared with the control (13.0%), noticeably greater after 3 h and when added to a high-cAMP medium (37.3% and 47.0% with E2 and P4, respectively). In conclusion, the results of this study demonstrate for the first time that ovine spermatozoa have progesterone and estrogen receptors and that both steroid hormones are related with the induction of the acrosome reaction.

A preliminary study of the effects of organic farming on oocyte quality in ewe lambs.

This study tested whether feeding Rasa Aragonesa ewes certified organic feed, from 15 days before mating until lamb weaning, improved oocyte quality and in vitro maturation (IVM) and fertilization (IVF) performances of the offspring. In a second experiment, ovaries from ewe lambs that were bred on an organic farm and were of the same breed were compared with those from conventionally bred animals. The number (± standard error of the mean) of healthy oocytes per ewe lamb did not differ significantly between organic (12.2 ± 3.3) and conventionally (13.6 ± 4.0) fed ewes. Ovaries from ewe lambs born on an organic farm had significantly (P < 0.0001) more healthy oocytes per ewe lamb (39.6 ± 5.2) than did those born on a conventional farm (25.0 ± 4.2), and higher IVM (76.5% vs. 53.1%, P < 0.0001) and IVF (97.3 vs. 91%, P < 0.05) rates. In conclusion, this preliminary approach to the study of the effect of organic procedures on the sheep oocyte quality indicates that the total integration in the complete organic system improved the oocyte quality of ewe lambs, although organic feeding alone was insufficient to improve quality.

Cleaved PARP-1, an Apoptotic Marker, can be Detected in Ram Spermatozoa.

The presence of apoptotic features in spermatozoa has been related to lower quality and functional impairment. Members of the poly-ADP-ribose polymerases (PARP) familyare involved in both DNA repair and apoptosis, playing important roles in spermatogenesis. Poly-ADP-ribose polymerase can be cleaved by caspases, and the presence of its cleavage product (cPARP) in spermatozoa has been related to chromatin remodelling during spermatogenesis and to the activation of apoptotic pathways. There are no reports on immunodetection of cPARP in ram spermatozoa; thus, we have tested a commercially available antibody for this purpose. cPARP was microscopically detected in the acrosomal ridge of some spermatozoa (indirect immunofluorescence). A preliminary study was carried out by flow cytometry (direct immunofluorescence, FITC). Ram semen was extended in TALP and incubated for 4 h with apoptosis inducers staurosporine (10 µm) or betulinic acid (200 µm). Both inducers and incubation caused a significant increase in cPARP spermatozoa (0 h, control: 21.4±3.3%, inducers: 44.3±1.4%; 4 h, control: 44.3±2.4%, inducers: 53.3±1.4%). In a second experiment, we compared the sperm fractions after density gradient separation (pellet and interface). The pellet yielded a slightly lower proportion of cPARP spermatozoa (28.5±1.2% vs 36.2±2.0% in the interface; p < 0.001), and a 12-h incubation increased cPARP similarly in both fractions (p < 0.001). cPARP seems to be an early marker of apoptosis in ram semen, although its presence in untreated samples was weakly related to worse quality (pellet/interface). We suggest to study the relationship of PARP and cPARP levels with between-male differences on sperm fertility.

Periconceptional undernutrition increases quantity and quality of oocyte population, but not cognitive or emotional response of 60-day-old lambs.

Maternal periconceptional undernutrition is associated with altered development and increased risks of adverse outcomes in the offspring. The aim of this work was to determine the effect of periconceptional undernutrition on behavioural and reproductive aspects of the offspring. Fifty ewes were synchronized in oestrus (day 0) and allocated to two groups (n = 25) to be fed diets that provided 1.5 (C) or 0.5 (L) times the requirements for maintenance until day 15. Ewes were mated and fed the control diet from day 16 until lambing. Two months after lambing, 26 lambs were exposed to tests to determine their cognitive/emotional responses. Six ewe lambs were euthanized and in vitro oocyte maturation and fertilization procedures performed. The experimental diets produced no changes of mean live weight (LW) of C ewes, L ewes presenting a reduction in their initial LW with significant differences at day 15, in comparison with C ewes (p < 0.05). L ewes experienced a significant reduction in their body condition (BC) in comparison with C ewes (p < 0.05). Fourteen days after the onset of the experimental diets, mean LW and BC of L ewes was significantly lower than those of C ewes (p < 0.05). Undernourished ewes presented a trend to a reduction of prolificacy and fecundity (p < 0.10) in comparison with C ewes. Emotional and cognitive test revealed a similar response between groups. Ewe lambs from the undernourished ewes presented a population of oocytes 1.7 times higher than ovaries from control ewe lambs (66.0 ± 0.73 vs. 113.7 ± 15.6 oocytes; p < 0.05) and had more oocytes in the 'good' (p < 0.05) and 'healthy' (p < 0.05) categories. In conclusion, a low plane of nutrition around conception significantly increases quantity and quality of the oocyte population of 60-day-old female descendants. Modifications of the cognitive and emotional responses of the progeny have not been evidenced.

Melatonin in sperm biology: breaking paradigms.

Melatonin is a ubiquitous molecule, present in a wide range of organisms, and involved in multiple functions. Melatonin relays the information about the photoperiod to the tissues that express melatonin-binding sites in both central and peripheral nervous systems. This hormone has a complex mechanism of action. It can cross the cell plasma membrane and exert its actions in all cells of the body. Certain melatonin actions are mediated by receptors that belong to the superfamily of G-protein-coupled receptors (GPCRs), the MT1 and MT2 membrane. Melatonin can also bind to calmodulin as well as to nuclear receptors of the retinoic acid receptor family, RORα1, RORα2 and RZRß. The purpose of this review is to report on recent developments in the physiological role of melatonin and its receptors. Specific issues concerning the biological function of melatonin in mammalian seasonal reproduction and spermatozoa are considered. The significance of the continuous presence of melatonin in seminal plasma with a fairly constant concentration is also discussed.

Proximate analysis and GC-MS phytochemical profiling of aqueous extracts of Doryopteris raddiana, a plant used by the Mbya-Guaraní as a contraceptive.

Doryopteris raddiana (Presl) Fée, a traditional contraceptive in Mbya culture, lacks scientific scrutiny regarding its chemical composition and contraceptive efficacy. Employing X-ray fluorescence, Fourier-transform infrared spectroscopy, and thermal analysis, we explored the plant's organs. Multielemental analysis excluded toxic elements. Key phytoconstituents identified by gas chromatography-mass spectrometry in the extracts obtained through infusion were glycerine, 1,3-dimethyl propane, and catechol in leaves; glycerine, cis-13-octadecenoic acid methyl ester, and 2-deoxy-D-erythro-pentose in stems and roots. Among these chemicals, glycerine emerged as the sole constituent with contraceptive potential, particularly intravaginally. Extract activity tests conducted on ram spermatozoa exhibited a reduction in the percentage of rapid spermatozoa but no significant impact on total motility, progressive motility, or viability. The reported data would only weakly support the advocated contraceptive action of this fern upon vaginal application, not through the oral administration of its decoction.

Involvement of progesterone and estrogen receptors in the ram sperm acrosome reaction.

The steroid hormones 17-ß estradiol (E2) and progesterone (P4) can regulate capacitation, hyperactive motility, and the acrosome reaction (AR) during the sperm transit through the female tract. Moreover, exogenous P4 and E2 can induce the AR in ovine spermatozoa, and progesterone receptor (PR) and estrogen receptors (ERα and ERß) are present in these cells. Thus, to investigate whether the effects both steroid hormones in ram sperm capacitation and AR are receptor-mediated, we incubated them with receptor agonists (tanaproget 1 µM and 5 µM for PR or resveratrol 5 µM and 10 µM for ER) or antagonists (mifepristone 4 µM and 40 µM for PR or tamoxifen 5 µM and 10 µM for ER) in capacitating conditions. The addition of receptor modulators did not affect sperm viability or total motility, although changes in progressive motility were detected. The incubation with both receptor agonists increased the percentage of acrosome-reacted spermatozoa, evaluated by chlortetracycline staining, when compared with the capacitated nontreated sample (Cap-C, P < 0.001). Moreover, the ER agonist resveratrol 10 µM provoked a greater AR than E2 (P < 0.01). Furthermore, the incubation with the receptor antagonists prevented the induction of the AR by P4 or E2, as the antagonists-treated spermatozoa presented a similar CTC pattern to that of Cap-C. In conclusion, these results confirm that P4 and E2 can induce the AR in ram spermatozoa and that this effect is receptor-mediated.

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.

Significance of non-conventional parameters in the evaluation of cooling-induced damage to ram spermatozoa diluted in three different media.

The objectives of this study were two. First, to compare three base media with different sugar composition as an initial step to achieve a good chemically-defined extender for ram sperm refrigeration. The second one, to determine which sperm quality parameters may be more useful for revealing differences between sperm samples. One medium contained 200 mM sucrose and 2.8 mM glucose (SM), another only disaccharides (D) such as sucrose, trehalose, maltose and lactose (75 mM each); and the third one (D+M) included a mix of monosaccharides (50 mM glucose, 20 mM fructose and 20 mM galactose,) and the same disaccharides as in D (50 mM each). Ram semen samples diluted in the mentioned media were refrigerated at 5°C for 1 h, and rewarmed upto 37°C in order to mimic the temperature in the female reproductive tract. Addition of monosaccharides to the extender did not produce a better preservation of motility or viability after cooling. The supplementation with other disaccharides apart from sucrose did not enhance the viability either. Thus, after cooling and rewarming, there were no significant differences in sperm viability (membrane integrity evaluated by CFDA/PI staining) or the percentage of progressive motile and rapid sperm (evaluated by CASA) between the three media. However, the percentage of viable non-capacitated sperm evaluated by the chlortetracycline (CTC) assay was higher and sperm oxygen consumption was lower in SM than in D and in D+M. Although the apoptosis-like markers [phosphatidylserine exposure assessed by Annexin V/CFDA staining and DNA-damage evaluated by TUNEL assay] showed a continuous increment throughout the process with all diluents, the percentage of sperm with damaged DNA at the end of the process was significantly lower in SM than in the other two media (p < 0.01). On the basis of these results, we would make two recommendations: the use of an extender supplemented only with sucrose and glucose for ram sperm refrigeration; the inclusion of non-conventional methods such as oxygen consumption measure, evaluation of capacitation state and apoptosis-like markers for revealing differences between sperm samples.

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.

Underlying molecular mechanism in the modulation of the ram sperm acrosome reaction by progesterone and 17ß-estradiol.

Steroid hormones progesterone (P4) and 17ß-estradiol (E2) not only have important functions in regulation of reproductive processes in mammals but also have direct effects on spermatozoa. There can be induction of the acrosome reaction in ram spermatozoa by P4 and E2 and, in the present study, there was further investigation of mechanisms underlying this effect. In a medium containing agents that increase cAMP, the presence of both P4 and E2 led to changes in the localization of proteins phosphorylated in tyrosine residues evaluated by indirect immunofluorescence. The inclusion of P4 at 1 µM in the media induced an increase in Ca2+i and mobilization in the area of the acrosome (Fluo-4 and Rhod-5 staining, respectively), an increase in ROS (H2DCFDA staining) and a substantial disruption of the acrosome (evaluated using RCA), while E2 did not have these effects. There were no effects on cAMP concentrations or PKA activity with inclusion of these hormones in the media. The inclusion of P4 at 100 pM in the media led to changes in values for sperm kinematic variables which could indicate there was an inhibition of the hyperactivation caused by agents that induce an increase in cAMP concentrations. In conclusion, results from the present study indicate that P4 and E2 promote mechanisms regulating the acrosome reaction in ram spermatozoa, however, these effects on mechanisms are different for the two hormones, and for E2, require further clarification.

The continuous presence of ewes in estrus in spring influences testicular volume, testicular echogenicity and testosterone concentration, but not LH pulsatility in rams.

The continuous presence of active male small ruminants prevents seasonal anestrus in females, but evidence of the same mechanism operating from the females to the males is scarce. This study assessed the effects of the continuous presence of ewes in estrus in spring on ram sexual activity, testicular size and echogenicity, and LH and testosterone concentrations. On 1 March, 20 rams were assigned to two groups (n = 10 each): isolated (ISO) from other sheep, or stimulated (STI) by 12 ewes, which were separated from the rams by an openwork metal barrier, allowing contact between sexes. Each week, four ewes were induced into estrus by intravaginal sponges. Live weight, scrotal circumference, testicular width (TW) and length (TL) were recorded at the beginning and at the end of the experiment, and testicular volume (TV) was calculated; at the same time, testicular ultrasonography and color Doppler scanning were performed. Blood samples (March to May) were collected once per week for testosterone determinations, and at the end of the experiment, blood samples were collected for 6 h at 20-min intervals for LH analysis. Rams were exposed to four estrous ewes in a serving-capacity test. Scrotal circumference, TW and TL were higher in the STI than in the ISO rams (P < 0.05) in May, and TV was higher (P < 0.05) in the STI (391 ± 17 cm3) than in the ISO rams (354 ± 24 cm3). In ISO rams, the number of white pixels was higher (P < 0.01) in May (348 ± 74) than in March (94 ± 21) and differed significantly (P < 0.01) from that of the STI rams in May (160 ± 33). In ISO rams, the number of grey pixels was higher (P < 0.05) in May (107 ± 3) than it was in March (99 ± 1). Stimulated and ISO rams did not differ significantly in mean LH plasma concentrations (0.8 ± 0.5 v. 0.9 ± 0.4 ng/ml), LH pulses (2.1 ± 0.5 v. 2.2 ± 0.2) and amplitude (2.0 ± 0.4 v. 3.2 ± 0.7 ng/ml, respectively). Stimulated rams had significantly higher testosterone concentrations than ISO rams from April to the end of the experiment. Stimulated rams performed more (P < 0.05) mountings with intromission (3.0 ± 0.4) than did ISO rams (1.5 ± 0.5). In conclusion, after 3 months in the continuous presence of ewes in estrus in spring, rams had higher TV and some testicular echogenic parameters were modified than isolated rams. Although exposed rams also had higher levels of testosterone after 2 months in the presence of estrous ewes, their LH pulsatility at the end of the study was not modified.

Polymorphisms of the melatonin receptor 1A (MTNR1A) gene influence the age at first mating in autumn-born ram-lambs and sexual activity of adult rams in spring.

The aim of this study was to determine whether polymorphisms of the melatonin receptor 1A (MTNR1A) gene influence the age at first mating in autumn-born ram-lambs and influence the out-of-season sexual activity of adult rams. In experiment 1, 24 Rasa Aragonesa ram-lambs born in September were genotyped for their RsaI and MnlI allelic variants of the MTNR1A gene, and the date of their first mounting with ejaculation after a period of semen collection training was documented. In experiment 2, the reproductive behavior, testicle size, and plasma testosterone concentrations of 18 adult rams (6 rams for each RsaI genotype) were recorded at the beginning (March) and end (May) of the seasonal anestrus. The number of days of training to achieve the first mating with ejaculation in T/T (C/C: 85.17 ± 12.08 C/T: 86.60 ± 18.87; T/T; 26.50 ± 24.50 d; P < 0.05), and G/G ram-lambs (G/G: 51.57 ± 14.99; A/G: 95.58 ± 10.95 d; P < 0.05) was significantly fewer than it was in the other genotypes. Likewise, for the RsaI genotype, 55% of the vulva-sniffing (P < 0.001), 48% of the approaches (P < 0.01), 48% of the mountings (P < 0.05) and 49% total activities (P < 0.001) were performed by T/T rams in March, and 50% of the sexual events in May (P < 0.001). For the Mnll variant, G/G rams performed a significantly (P < 0.001) larger proportion of the vulva-sniffing (41%), approaches (46%) and total activities (40%) in March, and 52% of the vulva-sniffing (P < 0.001), 43%, of the approaches (P < 0.001), 46% of the mountings (P < 0.05), and 47% of the total activities (P < 0.001) in May. Scrotal circumference, testicular volume, and plasma testosterone concentrations did not differ significantly among genotypes. Results confirmed that the polymorphisms of the MTNR1A gene sequence can influence reproductive performance in young and adult rams. Autumn-born ram-lambs that carried the T/T or G/G genotype had an advanced ability to reproduce, and T/T or G/G adult rams exhibited the most intense reproductive behavior. Genotyping might be a useful procedure for identifying the correct and rational use of rams in modern sheep farming.

Changes in calmodulin immunocytochemical localization associated with capacitation and acrosomal exocytosis of ram spermatozoa.

The aim of this study was to determine the localization of calmodulin (CaM) in ram sperm and the possible changes during in vitro capacitation (CA) and the ionophore-induced acrosome reaction (AR). Likewise, changes in intracellular calcium levels ([Ca(2+)](i)) were also analysed by using flow cytometry. CA was induced in vitro in a medium containing BSA, CaCl(2), NaHCO(3), and AR by the addition of the calcium ionophore A23187. The acrosomal status was assessed by the chlortetracycline-fluorescence (CTC) assay. Flow cytometry (FC) analyses were performed by loading samples with Fluo-3 AM, that emits fluorescence at a high [Ca(2+)](i), combined with propidium iodide (PI) that allowed us to discriminate sperm with/without an integral plasma membrane both with high/low [Ca(2+)](i). Immunocytochemistry localized CaM to the flagellum, and some sperm also contained CaM in the head (equatorial and post-acrosomal regions). CA and AR resulted in a slight increase in the post-acrosomal labelling. The treatment of sperm with increasing concentrations of two CaM antagonists, W7 and calmidazolium (CZ), accounted for an increase in capacitated and acrosome-reacted CTC-sperm patterns. CZ induced a significant reduction in the content of three protein tyrosine-phosphorylated bands of approximately of 30, 40 and 45kDa. However, W7 showed no significant effect at any of the studied concentrations. Neither of them significantly influenced protein serine and threonine phosphorylation. FC analysis revealed that the main subpopulation in the control samples contained 70% of the total sperm with integral plasma membrane and a medium [Ca(2+)](i). After CA, 67.1% of the sperm preserved an integral membrane with a higher [Ca(2+)](i). After AR, only 7.2% of the total sperm preserved intact membranes with a very high [Ca(2+)](i). These results imply that CaM appears to be involved in ram sperm capacitation, and both treatments increased its localization in the post-acrosomal region.

Effects of ageing and exogenous melatonin on pituitary responsiveness to GnRH in ewes during anestrus and the reproductive season.

The study examined the effect of melatonin implants on in vivo pituitary responsiveness to GnRH in control, fully productive (5.7+/-0.4 years old, n=17) and aged (10.7+/-0.3 years old, n=14) ovariectomized, estradiol-treated Rasa Aragonesa ewes. On 27 February, eight ewes in each age group received a single implant containing 18 mg melatonin. On 10 April, blood samples to be assayed for LH were collected at 10-min intervals over 4h (starting at 09:00 and 22:00 h). After samples 6 and 18 were collected, ewes received a single i.v. injection of GnRH (20 ng/kg liveweight). The pituitary response to GnRH was assessed using the difference between plasma LH concentrations before and after (highest value) each injection (DLH1, DLH2)), and the area under the LH response curve for 1h after each GnRH injection (AUC1, AUC2). On 23 September, the previously implanted ewes received a new melatonin implant and, on 17 November, all of the ewes were subjected to the same diurnal and nocturnal sampling protocols, again. Generally, non-implanted aged ewes exhibited a lower pituitary response to GnRH than did non-implanted control ewes, particularly in November and after the first injection (P<0.05 for DLH1 and AUC1 in both the diurnal and nocturnal tests). The response was significantly affected by the interaction of age and melatonin treatment, particularly in the diurnal tests (P<0.1 for DLH1 and AUC1, and P<0.05 for AUC2 in April; P<0.05 for DLH1, AUC1 and AUC2 in November), which indicated that exogenous melatonin increased LH levels after GnRH injections in aged ewes compared to non-implanted ewes, this effect being the opposite in control females. Thus, melatonin can restore in ewes the functionality of the neuroendocrine system, after it has been reduced by senescence.

Profile and reproductive roles of seminal plasma melatonin of boar ejaculates used in artificial insemination programs.

Melatonin (MLT) is present in seminal plasma (SP) of mammalian species, including pigs, and it is credited with antioxidant properties. This study aims to identify the sources of variation and the role of boar SP MLT on sperm quality and functionality and in vivo fertilizing ability of liquid-stored semen doses used in AI programs. The SP MLT was measured using an ELISA kit in a total of 219 ejaculates collected from 76 boars, and reproductive records of 5,318 AI sows were recorded. Sperm quality was assessed according to motility (computer-aided sperm analysis) and viability (cytometry evaluation). Sperm functionality was assessed according to the cytometric determination of intracellular HO generation, total and mitochondrial O production, and lipid peroxidation in liquid AI semen samples stored at 17°C over 144 h. The concentration of SP MLT differed among seasons ( < 0.01) and day length periods ( < 0.001) of the year, demonstrating that the ejaculates collected during the increasing day length period (9.80 ± 1.38 pg/mL, range: 2.75-21.94) had lower SP MLT concentrations than those collected during the decreasing day length period (16.32 ± 1.67 pg/mL, range: 5.02-35.61). The SP MLT also differed ( < 0.001) among boars, among ejaculates within boar, and among portions within the ejaculate, demonstrating that SP from the first 10 mL of sperm-rich ejaculate fraction (SRF) exhibited lower MLT concentrations than post-SRF. The SP MLT was negatively related ( < 0.001) to mitochondrial O production in viable sperm. The SP MLT did not differ among AI boars ( = 14) hierarchically grouped according to high and low fertility outcomes. In conclusion, SP MLT concentration in AI boars varies depending on the season of ejaculate collection and differs among boars, ejaculates within boar, and portions within ejaculate. The SP MLT may act at the mitochondrial level of sperm by reducing the generation of O. However, this antioxidant role of SP MLT was not reflected in sperm quality or in vivo fertility outcomes of AI semen doses.

The effect of melatonin implants during the seasonal anestrus on embryo production after superovulation in aged high-prolificacy Rasa Aragonesa ewes.

The aim of this study was to assess the effect of melatonin implants administered in March on the ovarian cyclicity, ovulatory response and embryo production after repeated superovulation of selected high-prolificacy Rasa Aragonesa aged ewes. During the seasonal anestrus of two consecutive years, 113 superovulatory treatments have been performed. Ewes were treated (M) or not (C) with melatonin implants in March (day 0). All of them received intravaginal progestogen sponges on day 24 (recovery 1) and 80 (recovery 2) after melatonin implants insertion in year 1, and on day 28 and 77 in year 2. The intravaginal sponges were removed after 14 days. Superovulatory treatments consisted of eight doses in decreasing concentrations (2 mL x 2 and 1 mL x 6) of oFSH (Ovagen) administered twice daily starting 72 h before sponge removal. Seven days after the onset of estrus, embryos were recovered by laparotomy. Melatonin increased cyclicity only in recovery 2 year 2 (83% versus 42%; P < 0.05) but not in the other experimental periods. Among the 78% (88) ewes that ovulated and produced functional corpora lutea, melatonin implants tended to improve embryo viability in recovery 2 by increasing the number of blastocysts per superovulatory treatment (2.4 +/- 0.6 versus 1.1 +/- 0.4; P = 0.09), the rate of viability (67 +/- 9% versus 43 +/- 9%; P < 0.05), and freezability (55 +/- 9% versus 33 +/- 8%; P < 0.05). More specifically, melatonin induced a significant reduction of the number and rate of non-viable (degenerate and retarded) embryos in recovery 2 (0.4+/-0.1 embryos versus 1.3 +/- 0.3 embryos and 4 +/- 1% versus 22 +/- 6%, respectively; P < 0.05). Our results demonstrate that melatonin implants in March can improve at medium term (3 months after implantation) the viability of embryos collected from selected high-prolificacy Rasa Aragonesa aged ewes after superovulation.

Expression, cellular localization, and involvement of the pentose phosphate pathway enzymes in the regulation of ram sperm capacitation.

Spermatozoa require substantially more ATP than other cells, not only for sustaining sperm motility but also for regulating protein phosphorylation during capacitation. In this study, we have reported for the first time the presence of the two key enzymes of the pentose phosphate pathway (PPP), glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in ovine spermatozoa by indirect immunofluorescence, Western blotting, in-gel activity, and reverse transcription polymerase chain reaction analysis. We found that the activity of both enzymes significantly increased after in vitro capacitation in the presence of high-cAMP levels, with a concomitant increase in protein tyrosine phosphorylation and in the proportion of sperm-capacitated pattern assessed by the chlortetracycline staining. These results suggest that PPP is related with the progress of capacitation and that a relationship between calcium compartmentalization, protein tyrosine phosphorylation and PPP seems to exist. This is the first report that shows a connection between the PPP, cAMP/PKA signaling pathways and sperm capacitation. These findings can be of high-biological importance to improve our knowledge of the biochemical mechanisms involved in the acquisition of mammalian sperm functional competence and, ultimately, fertility.

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.

Ram seminal plasma proteins contribute to sperm capacitation and modulate sperm-zona pellucida interaction.

Incubation of ram spermatozoa in capacitating conditions with cAMP-elevating agents promotes a progressive time-dependent increase in the capacitated sperm subpopulation. In this study, the fertilizing capacity of ram spermatozoa (ability to bind to the zona pellucida, ZBA rate) capacitated in these conditions was determined. The results showed an increase (P < 0.001) in ZBA rate related to control samples in basal medium that contained BSA, calcium, and bicarbonate (1.97 ± 0.19 vs. 1.31 ± 0.09 sperm bound/oocyte, respectively). A significant correlation between protein tyrosine phosphorylation and ZBA rate (P < 0.05, r = 0.501) corroborated that incubation in a "high-cAMP" environment improves the fertilizing ability of ram spermatozoa. Likewise, the presence of two seminal plasma (SP) proteins able to protect sperm against cold shock (RSVP14 and RSVP20) was evidenced in both SP and the ram sperm surface, and their influence in the fertilizing ability of spermatozoa capacitated in basal medium or with cAMP-elevating agents was determined. The results verified that RSVP14 and RSVP20 act as decapacitating factors given that their addition to SP-free sperm samples previously to capacitation maintained high proportions of the noncapacitated sperm pattern with no increase in protein tyrosine phosphorylation. However, the obtained ZBA rate in the high-cAMP-containing samples was increased in the presence of RSVP20 (P < 0.05). These findings would indicate that the stimulating effect exerted by this protein on the sperm-oocyte binding occurs downstream from the cAMP generation and that the mechanisms by which RSVP20 promotes the zona pellucida binding might be independent of protein tyrosine phosphorylation.