L.A. Zarazaga

Effect of level of dietary energy and protein on embryo survival and progesterone production on day eight of pregnancy in Rasa Aragonesa ewes

The aim of this experiment was to investigate the effects of dietary protein and energy on ovulation rate and embryo survival to day 8 of pregnancy, and the associated concentrations of progesterone in jugular, ovarian and uterine veins, in a Spanish breed of sheep. In mid-October, three groups of ewes were fed to provide 1.5 x (H; n = 9), 0.5 x (L; n = 12) or 0.5 x plus 7.44 g CP/MJ ME (LP; n = 8) energy requirements for maintenance of live weight from day -14 relative to a synchronized mating on day 0. A significant effect of nutrition on ovulation rate was observed (H: 2.22 +/- 0.16; L: 1.50 +/- 0.16; LP: 1.88 +/- 0.12 corpora lutea; P < 0.05). Mean LH and progesterone concentrations were affected by nutrition on day 7, L ewes showing the highest mean LH level (P < 0.01), while H ewes presented the lowest mean LH concentration and the highest mean plasma progesterone concentration (P < 0.01). Laparotomies were performed on six animals of each group on day 8 to determine the effect of nutrition on embryo development. A significantly higher percentage of embryos recovered from L and LP ewes presented an earlier stage of development (morulae or early blastocysts) (P < 0.001), while 100% embryos of H ewes were expanded blastocysts. The ratio expanded blastocysts/corpora lutea was significantly higher in H ewes (0.86) when compared with L and LP groups together (0.57; P < 0.05). Mean progesterone concentration in the ovarian vein was 800-fold higher than mean jugular venous levels with no differences between groups. Samples from ovarian veins contralateral to corpus luteum-bearing ovaries showed mean progesterone concentrations significantly lower than samples opposite to corpus luteum (ipsilateral: 1037.84 +/- 138.45; contralateral: 30.4 +/- 11.22 ng/ml; P < 0.001). Mean progesterone concentration in the uterine vein was approximately 30-fold higher than in jugular and similar in both uterine horns and treatments. No effect of nutrition on pregnancy rate was observed (H: 89%; L: 92%; LP: 100%). These results suggest that neither dietary energy nor protein are able to modify pregnancy rate or progesterone concentrations in ovarian and uterine veins eight days after mating. However, the delay in embryo development observed in the embryos collected from L and LP ewes may give rise to compromised embryo growth and development some days later.

Effect of exogenous melatonin and plane of nutrition after weaning on estrous activity, endocrine status and ovulation rate in Salz ewes lambing in the seasonal anestrus

Forty-nine Spanish Salz ewes lambing in the second fortnight of March (20 March +/- 1.5 d) were used to determine the effects of exogenous melatonin and postweaning nutrition on endocrine status, date of first estrus and ovulation rate. Experimental design was a factorial defined by 2 postweaning planes of nutrition, 1.80 (high) and 1.35 (low) times the maintenance requirements, and treatment with a single 18-mg subcutaneous implant of melatonin (M) 32 d after lambing or no treatment control (C). Mean weaning to first estrus interval was shorter in treated than in control ewes (50.8 +/- 4.2 vs 87.6 +/- 6.3 d; P < 0.01). Considering both the treated and control animals together, the ratio between mean night and daytime plasma melatonin levels was significantly correlated with the implant insertion-first estrus interval on Day 5 (0.67; P < 0.01) and Day 35 (0.63; P < 0.05) after implantation. Melatonin implants induced a significant increase of mean LH concentrations at Days 14 and 33 after implantation (P < 0.01) without any significant influence of plane of nutrition. Ovulation rate was higher for treated than control ewes in the second estrus (P < 0.05). An interaction between plane of nutrition and exogenous melatonin on ovulation rate at the second cycle after weaning was detected (P < 0.01), being close to the significance in the first, fourth and fifth cycles (P < 0.1). These results suggest that exogenous melatonin in April may be an effective way of advancing the breeding season and enhancing ovulation rate associated with a low rather than a high plane of nutrition.

Effect of undernutrition on the distribution of progesterone in the uterus of ewes during the luteal phase of the estrous cycle

The effect of undernutrition on ovarian and uterine venous progesterone concentrations and endometrial progesterone content on Days 5 and 10 of the estrous cycle were studied. Forty ewes were synchronized using progestagen pessaries. At pessary withdrawal, the ewes were fed diets to provide either 1.5 or 0.5 times the daily maintenance requirement (Group H, n = 20 and Group L, n = 20, respectively). Ewes fed the low nutrition diet (Group L) had higher mean peripheral progesterone concentrations than those fed the high plane diet (Group H; P < 0.05) but lower endometrial progesterone content on Day 5 (P < 0.05). Neither ovarian nor uterine venous levels were affected by nutrition on either Day 5 or 10. Progesterone concentrations in blood samples collected ipsilateral to ovaries bearing a corpus luteum (CL) were higher than in the contralateral samples (P < 0.001). It is concluded that undernutrition can produce a reduction of endometrial content of progesterone the first week after mating. Since no differences in ovarian venous concentrations were observed, it remains to be shown whether this variation is due to other variables, such as the population of endometrial progesterone receptors or other nonhormonal factors.

Effects of season and feeding level on reproductive activity and semen quality in Payoya buck goats

The aim of this study was to determine if there is a seasonal pattern of reproductive activity in male Payoya goats and if this seasonality can be modulated by a higher level of nutrition. For a period of 16 months, 10 adult bucks were divided into two experimental groups that differed in their feeding level. The high nutrition group (H, n=5) received 1.6 times their maintenance food requirements. The control nutrition group (C, n=5) received a diet that supported 1.1 times their maintenance requirements. Body weight and testosterone concentrations were determined weekly, and testicular weight was determined every 2 weeks. Sexual behaviour and semen characteristics were determined monthly. Feeding level did not affect the onset or the end of the reproductive activity as measured by testosterone concentrations, with high testosterone concentrations between July and November. Ejaculation latency was positively influenced by feeding level: 43.2+/-2.2s vs. 61.6+/-3.2s for H and C group, respectively (P<0.001). The percentage of males that ejaculated or that were sexually active was higher in the H group (P<0.01). No differences between feeding levels were observed in the different semen characteristics studied. However, major differences between months were observed for all studied variables. These results lead us to conclude that Payoya bucks exhibit large seasonal variation in their reproductive activity. Higher feeding level allowed a better sexual behaviour in bucks in late spring, when male effect is used on the local livestock to breed females.

Effect of melatonin implants on sexual activity in Mediterranean goat females without separation from males

This work was designed to determine whether melatonin treatment at the spring equinox can induce reproductive activity in goats without separation from males (separation being the normal practice in Spanish farming systems) and whether this treatment modifies the onset of the natural breeding season. Twenty-nine entire does were distributed into two groups (Group M, n=14; Group C, n=15). A third group of ovariectomized, estradiol-treated goats (OVX group, n=5) was used to study the effect of melatonin on reproductive activity. On March 18, Groups M and OVX received a subcutaneous melatonin implant. In entire females, estrus was tested daily using entire aproned males, and ovulation rate was assessed after identification of estrus. Plasma progesterone in entire goats, plasma luteinizing hormone (LH) in the OVX group, and live weight and body condition score for all animals were recorded once a week. In entire goats, a clear treatment by time interaction was observed for progesterone concentrations (P<0.001), with a period of high progesterone concentrations during the natural seasonal anestrus in Group M. A similar period of high LH concentrations was observed in the OVX group. Whereas all females of Group M presented ovarian activity during this period, no female of Group C did. The resumption of the natural breeding season was retarded in Group M in comparison with that in Group C (P<0.05). We can conclude that in Mediterranean goats, melatonin implants can induce reproductive activity without separation from males, and it causes a small retardation in the reactivation of reproductive activity in the natural breeding season.

The large variability in melatonin blood levels in ewes is under strong genetic influence

The present study was conducted to assess the degree of genetic determination of the variability in the mean nocturnal plasma concentration of melatonin in sheep. Three hundred twelve ewes born from 18 males and with known genealogy were sampled at the summer and the winter solstices. The nocturnal plasma melatonin concentration was defined as the mean of four plasma samples taken at hourly intervals in the middle of the night (2200-0200). Identity of the father ( P < 0.001) and the solstice ( P < 0.05) were significant. Melatonin concentrations varied considerably among individuals [338.4 ± 197.5 (SD) pg/ml; range 26.6-981.3 pg/ml] and between rams regarding the melatonin concentrations of their daughters (range from 202.9 to 456.3 pg/ml). Inheritance was analyzed by a statistical model that allows discrimination of genetic effects from nongenetic effects and that estimates repeatability and heritability coefficients. Both the repeatability coefficient between solstices (0.60) and heritability coefficient [0.45 ± 0.07 (SE)] were high. These results demonstrate that the variability in plasma melatonin concentration in ewes is under strong genetic control.The present study was conducted to assess the degree of genetic determination of the variability in the mean nocturnal plasma concentration of melatonin in sheep. Three hundred twelve ewes born from 18 males and with known genealogy were sampled at the summer and the winter solstices. The nocturnal plasma melatonin concentration was defined as the mean of four plasma samples taken at hourly intervals in the middle of the night (2200-0200). Identity of the father (P < 0.001) and the solstice (P < 0.05) were significant. Melatonin concentrations varied considerably among individuals [338.4 +/- 197.5 (SD) pg/ml; range 26.6-981.3 pg/ml] and between rams regarding the melatonin concentrations of their daughters (range from 202.9 to 456.3 pg/ml). Inheritance was analyzed by a statistical model that allows discrimination of genetic effects from nongenetic effects and that estimates repeatability and heritability coefficients. Both the repeatability coefficient between solstices (0.60) and heritability coefficient [0.45 +/- 0.07 (SE)] were high. These results demonstrate that the variability in plasma melatonin concentration in ewes is under strong genetic control.

Seasonal changes in melatonin concentrations in female Iberian red deer (Cervus elaphus hispanicus)

Abstract: In deer, most of the earlier investigations on pineal function examined the effects of artificial photoperiods or the administration of melatonin to manipulate reproduction. However, endogenous melatonin rhythms have not been studied in red deer. Thus, we monitored seasonal changes in plasma melatonin concentrations in 16 adult female Iberian red deer living in outdoor enclosures. Blood was sampled on the day of each seasonal change every 3–4 hr overnight and 1 hr before and after sunset and sunrise. In addition, in six of the previous hinds, blood sampling during the hour prior and after sunset and sunrise was collected every 20 min. Significant differences were found both in amplitude and duration of the nocturnal plasma melatonin profiles in the four seasonal changes (P<0.01). The nocturnal mean level of melatonin, the duration of nocturnal secretion levels and maximal concentrations were significantly higher at the winter solstice than in summer solstice or equinoxes (P<0.05). Moreover, the mean overnight concentrations were significantly higher at the spring equinox and winter solstice than during the summer solstice and autumn equinox (P<0.05). A pronounced elevation from low levels was recorded 1 hr after sunset, remained elevated during the hours of darkness and declined to low levels 1 hr after dawn. Concentrations close to sunrise were higher than those near sunset at all changes of season (P<0.05). These results show for the first time in red deer that the pineal gland of the adult female is highly responsive to both daily and seasonal changes in natural environmental illumination, although overnight levels lasted longer than the photoperiodic night is all cases, particularly at the winter solstice.

Genetic variability in plasma melatonin in sheep is due to pineal weight, not to variations in enzyme activities.

This study was conducted to determine the origin of the high variability in the mean nocturnal plasma melatonin concentration (MC) in sheep. Two extreme groups of 25 lambs each [low (L) and high (H)] were obtained by calculating their genetic value on the basis of the MC of their parents. The MC of lambs was significantly higher in the H group than in the L group (L: 189.7 ± 24.4 vs. H: 344.1 ± 33.0 pg/ml, P < 0.001). Within each group, 13 lambs were slaughtered during the day (D) and 12 lambs during the night (N). Pineal weight was significantly higher in the H group than in the L group (L: 83.5 ± 6.7 vs. H: 119.1 ± 9.2 mg, P < 0.01) but did not differ between D and N. The amount of melatonin released in vitro per milligram of pineal gland, the arylalkylamine N-acetyltransferase (AANAT) activity, the AANAT protein content, and the level of AANAT mRNA differed significantly between D and N but not with genetic group. Hydroxyindole O-methyltransferase activity did not differ significantly between D and N or between genetic groups. Therefore, the genetic difference in MC between the two groups of lambs was attributed to a difference in pineal size, not in enzymatic activity of the pinealocytes.

Genetic variability in melatonin concentrations in ewes originates in its synthesis, not in its catabolism

We investigated whether the genetic difference in plasma melatonin concentration in ewes was due to differences in the synthesis pathway from the pineal gland or in the catabolism of the hormone. Two groups of ewes [9 low (L) and 10 high (H)] were selected according to the breeding value of their mean nighttime plasma melatonin concentrations estimated at winter and summer solstices. In response to an identical dose of melatonin administered intravenously at 9:00 AM, no differences between groups were observed for any of the kinetic parameters: clearance rate, steady-state volume of distribution, terminal half-lives, and mean residence times. In the second experiment, two series of frequent blood samples were performed, one in the middle of the dark phase with samples taken every 5 min, and the other over 24 h with hourly samples. Highly significant differences between groups in nocturnal melatonin production rate were observed (L: 25.7 ± 2.8 vs. H: 63.1 ± 8.9 μg ⋅ kg-1 ⋅ h-1, P < 0.01). Thus the genetic differences in plasma melatonin concentrations in ewes originate in the synthesis pathway of the melatonin from the pineal gland rather than from differences in the catabolism of the hormone.We investigated whether the genetic difference in plasma melatonin concentration in ewes was due to differences in the synthesis pathway from the pineal gland or in the catabolism of the hormone. Two groups of ewes [9 low (L) and 10 high (H)] were selected according to the breeding value of their mean nighttime plasma melatonin concentrations estimated at winter and summer solstices. In response to an identical dose of melatonin administered intravenously at 9:00 AM, no differences between groups were observed for any of the kinetic parameters: clearance rate, steady-state volume of distribution, terminal half-lives, and mean residence times. In the second experiment, two series of frequent blood samples were performed, one in the middle of the dark phase with samples taken every 5 min, and the other over 24 h with hourly samples. Highly significant differences between groups in nocturnal melatonin production rate were observed (L: 25.7 +/- 2.8 vs. H: 63.1 +/- 8.9 microg . kg-1 . h-1, P < 0.01). Thus the genetic differences in plasma melatonin concentrations in ewes originate in the synthesis pathway of the melatonin from the pineal gland rather than from differences in the catabolism of the hormone.

Oestrous activity, ovulation rate and plasma melatonin concentrations in Rasa Aragonesa ewes maintained at two different and constant body condition score levels and implanted or reimplanted with melatonin

Forty-seven adult and non-pregnant Rasa Aragonesa ewes were used to study the effect of two constant body condition score levels on reproductive parameters in ewes implanted in early April or reimplanted 49 days later with melatonin. The duration of the experiment was 9 months (27 November 1992 – 3 September 1993), and was designed as a factorial study defined by two constant body condition (BC) scores, high (H; 2.75 or greater) or low (L; 2.50 or less) and the implant on 8 April (M) or reimplant (2M) with melatonin (18 mg, Melovine™). Oestrus was detected daily using vasectomized rams and ovulation rate was measured by laparoscopy 6 days after positive identification. Blood samples were collected twice weekly from each ewe and assayed for progesterone. In addition, samples were collected from 20 ewes (five in each treatment group) every 2 h and hourly for 24 h on Days 27 and 84 after first implantation, respectively, and assayed for melatonin. n nOnset of seasonal anoestrus was significantly influenced by the BC score (February 16 ± 8 and March 15 ± 5 for H and L groups; P < 0.01), but neither of the factors considered in the study influenced the interval between the first implantation (8 April) and the first detected oestrus (54 ± 7 days, 63 ± 8 days, 64 ± 10 days and 66 ± 5 days for HM, LM, H2M and L2M groups, respectively). Ovulation rate in the third cycle detected from 1 month after first implant insertion was significantly influenced by the BC score level (1.78 vs. 1.44 corpora lutea for H and L groups; P < 0.05). However, ovulation rate in the first and second cycles was slightly lower in the H group (1.46 vs. 1.64 and 1.48 vs. 1.56 corpora lutea, respectively), suggesting a positive effect in the short term of the melatonin implants in ewes with a moderately low BC score. No significant effect of the reimplant on ovulation rate was detected. Plasma melatonin concentrations on Day 84 were not different between groups of ewes receiving one or two implants. Furthermore, and except for nocturnal levels in the high BC score group, ewes that received one implant showed significantly higher melatonin concentrations on Day 84 than on Day 27 after implantation. It is concluded that (i) interval between melatonin implantation and first oestrus is not influenced by the BC score level; (ii) the reimplant does not improve reproductive parameters, although exogenous melatonin seems to overcome the positive effect of a high BC score in the early breeding season; (iii) one implant is adequate to maintain high plasma melatonin levels up to 3 months after implantation.