F. Forcada

Seasonal changes in oestrus activity and ovulation rate in Rasa Aragonesa ewes maintained at two different body condition levels

Abstract Onset and duration of seasonal anoestrus and seasonal changes in ovulation rate were studied for two consecutive years in two groups of Spanish Rasa Aragonesa ewes with body condition (BC) scores ⩽ 2.5 (low BC group) or ⩾ 2.75 (high BC group). Animals were kept penned throughout the experiments (November 1987–September 1988 and February–September 1989) (latitude 41° 40′N). Mean liveweights and BC were similar at the beginning and end of both study periods. Oestrus was tested daily using aproned males. Ovulation rate was measured by laparoscopy 6 d after identification of oestrus. Animals were blood sampled for progesterone determination every 6 d during the 1989 experiment. The high BC group of ewes showed a shorter seasonal anoestrus than the low BC group in both study years (64 vs. 113 and 76 vs. 92 d in 1988 and 1989) ( P P P P

The effect of melatonin on the secretion of progesterone in sheep and on the development of ovine embryos in vitro.

Two experiments were carried out in order to determine whether melatonin can improve secretion of progesterone in vivo, and its effect on embryonic development in vitro. In the first experiment, blood samples were collected from 5 ewes at 15 min intervals for 2 h at 7 and 10 days after withdrawal of progestagen pessaries. The first hour constituted a control period, which ended with an intravenous administration of 3 μg/(kg bw)0.75 melatonin. All the ewes on day 7 and three of the ewes on day 10 showed a progesterone response to melatonin challenge, defined as an increase in the plasma progesterone concentration in at least two consecutive samples during the post-treatment period above the mean+2SD of the values in the pre-treatment period. A paired t-test revealed a significant effect of melatonin on the overall plasma progesterone concentrations before and after the challenge, both on day 7 (pre, 0.61±0.11; post, 0.73±0.13 ng/ml; p<0.01) and day 10 (pre, 1.16±0.19; post, 1.30±0.20 ng/ml; p<0.05). Ninety-one thawed embryos (46 morulae and 45 blastocysts) were used in the second experiment, being cultured with or without 1 μg/ml melatonin. If the embryos were blastocysts when the culture started, melatonin increased the percentage that had hatched after 24 h of culture (p<0.01), and there was a lower percentage of degenerated embryos at the end of the incubation period (p<0.05). It may be concluded that melatonin treatment in sheep can increase both fertility and prolificacy by improving luteal function and embryonic survival.

Hormonal control of reproduction in small ruminants.

Reproduction of small ruminants can be controlled by several methods developed in recent decades. Some of these involve administration of hormones that modify the physiological chain of events involved in the sexual cycle. Methods which utilise progesterone or its analogues are based on their effects in the luteal phase of the cycle, simulating the action of natural progesterone produced by the corpus luteum after ovulation, which is responsible for controlling LH secretion from the pituitary. Use of prostaglandins is an alternative method for controlling reproduction by eliminating the corpus luteum and inducing a subsequent follicular phase with ovulation. Finally, the discovery of the properties of melatonin in photoperiod-dependent breeding animals opened up a new methodology to control reproduction in these species, inducing changes in the perception of photoperiod and the annual pattern of reproduction. Use of hormones to induce oestrus has allowed increased use of artificial insemination in small ruminants, a very useful management tool, considering the difficulty of detecting oestrus in these species. At commercial level, synchronisation of oestrus allows control of lambing and kidding, with subsequent synchronisation of weaning of young animals for slaughter. Also, it allows more efficient use of labour and animal facilities. Multiple ovulation and embryo transfer programmes are also possible with the use of oestrus synchronisation and artificial insemination. Finally, hormonal treatments have also been used to induce puberty in ewe-lambs and doelings.

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.

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.

Pharmaceutical Control of Reproduction in Sheep and Goats

Small ruminant species such as sheep and goats are short-day breeders, which is a crucial factor affecting the offer of lambs and kids throughout the year. An appropriate management of reproduction allows ewes and does to breed in the spring to increase the supply of product to the marketplace on a year-round basis. Pharmaceutical control of reproduction is possible, usually through administration of hormones or analogues related to the natural estrous cycle, such as progesterone, prostaglandins, and/or melatonin.

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.

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.

Effect of undernutrition on uterine progesterone and oestrogen receptors and on endocrine profiles during the ovine oestrous cycle

In the present study, it was investigated whether undernutrition affected the binding capacity, immunoreactivity and mRNA expression for uterine oestrogen and progesterone receptors (ER and PR, respectively) in sheep, as well as whether the responses were associated with changes in plasma concentrations of progesterone (P4), oestradiol (E2), glucose, fatty acids, insulin, leptin and insulin-like growth factor (IGF)-I during the oestrous cycle. Twenty ewes were fed either 1.5 (C) or 0.5 (L) times their maintenance requirements and were killed on Day 5 or 14 of the cycle (Day 0 = oestrus). Compared with Group C, Group L had higher concentrations of non-esterified fatty acids and lower concentrations of insulin, leptin and IGF-I. Group L also had higher plasma concentrations of P4 during the final days of the luteal phase. At oestrus in both treatment groups, there were peaks in the concentrations of glucose, insulin and IGF-I. For ER and PR, transcript expression, binding capacity and immunoreactivity were higher on Day 5 than on Day 14 of the cycle. The binding capacities for ER and PR were lower in Group L than in Group C on Day 5. Group C showed more immunoreactive staining for ER than did Group L in two of five cell types, whereas no effect of treatment was observed for PR immunoreactivity. There was more PR mRNA in the uterine horn contralateral to the corpus luteum in Group C than in Group L ewes. We conclude that undernutrition impairs steroid receptor expression and binding capacity. This may alter the uterine environment and help explain the reductions in embryo survival.

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.