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.     

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 × 2 × 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.     

Melatonin membrane receptors MT1 and MT2 are expressed in ram spermatozoa from non-seasonal breeds

Tropical Animal Health and Production - In mammals, many melatonin biological functions are mediated through its interaction with the membrane receptors MT1 and MT2. We have previously reported...     

Presence of melatonin‐catabolizing non‐specific enzymes myeloperoxidase and indoleamine 2,3‐dioxygenase in the ram reproductive tract

The melatonin catabolism is very complex and not completely understood. Melatonin can be metabolized by free radical interaction, but also pseudo‐enzymatically or by enzymatic pathways. We have previously detected the existence of melatonin‐synthesizing enzymes and melatonin receptors MT1 and MT2 in the ram reproductive tract; thus, in order to start to elucidate melatonin catabolism in these organs, we have investigated the presence of the melatonin‐catabolizing enzymes indoleamine 2,3‐dioxygenase (IDO, both IDO1 and IDO2 isoforms) and myeloperoxidase (MPO) in testis, epididymis and accessory glands. Gene expression analyses by real‐time PCR showed the presence of MPO, IDO1 and IDO2 in all the organs of the ram reproductive tract and revealed that MPO is the main melatonin‐catabolizing enzyme, which is mainly expressed in the testis and the bulbourethral glands (p < .05). These results were further corroborated by immunohistochemical staining, and by Western blot. Likewise, MPO was also evidenced in epididymal and ejaculated spermatozoa by indirect immunofluorescence and Western blot. In conclusion, melatonin‐catabolizing enzymes MPO, IDO1 and IDO2 are expressed in the ram reproductive tract, and MPO is the most expressed one, mainly in the testis and the bulbourethral glands. The presented results warrant further studies on the function of these enzymes and their melatonin‐metabolizing activity.     

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.