Dietary restriction in sheep: Uterine functionality in ewes with different body reserves during early gestation.

The aim of this study was to characterize insulin, leptin and IGF-1 profiles after undernutrition in pregnant ewes with different initial body condition (iBCS) and to investigate embryo quality, uterine gene expression and presence and location of proteins during early gestation (day 5). Thirty-six Rasa Aragonesa ewes were divided into 2 groups with different BCS: BCS> 2.75 (high, H, n = 19) and BCS <2.25 (low, L, n = 17) and they were randomly assigned to two nutritional treatments: 1.5 maintenance (M) (control, C) or 0.5 (M) (undernourishment, U) times the daily maintenance requirements establishing four groups: high-iBCS control (HC, n = 9), high-iBCS undernourished (HU, n = 10), low-iBCS control (LC, n = 9) and low-iBCS undernourished (LU, n = 8). High-iBCS ewes presented higher concentration of IGF-1, reflecting a better metabolic status in these animals. There was a greater proportion of high-iBCS ewes presenting more than one CL (P < 0.05), and associated greater P4 plasma concentration, number of recovered embryo and a tendency for higher embryo viability rate (P = 0.13). In uterus, undernourished ewes tended to present lower P4 (P = 0.09) and higher E2 concentration (P = 0.10). Inmunostaining of uterine progesterone and estrogen receptors (PR and ERα) was not affected by iBCS and nutritional treatment. Ewes with low-iBCS tended to have more INSR mRNA, and undernourished ewes tended to have more IGFBP2 mRNA expression (P < 0.08). An interesting finding was that the uterine response to undernutrition was dependent on iBCS: a higher expression of GHR (P < 0.05) and a tendency in IGFBP5 (P = 0.09) mRNA was found in undernourished than control ewes but only in the high-iBCS group. In summary, the present study demonstrates that the endocrine response and the uterine gene expression to undernutrition depend on the initial body energy reserves (iBCS) and appears to be associated with a differential embryo quality.

Presence of photoperiod-melatonin-induced, sexually-activated rams in spring advances puberty in autumn-born ewe lambs.

The objective of this study was to determine the effect of sexually activated (by specific photoperiodic treatments) vasectomized rams on the onset of puberty in autumn-born ewe lambs in spring. Photoperiodic-treated rams were assigned to one of three groups, which were sequentially exposed to two months of long days (16h light/d) and given three melatonin implants at the end of the long days (sexually-activated-rams; SAR): SAR1 (n=5; 1 December-31 January), SAR2 (n=5; 1 Jan-28 February), and SAR3 (n=5; 1 February-31 March). Control rams (CR; n=4) were exposed to the natural photoperiod. On 1 March, 50 ewe lambs born in September were assigned to the SAR rams (SAR-treated; n=25) or the CR rams (CR-treated; n=25). SAR-treated ewe lambs were housed with SAR1 rams from 1 March. SAR2 rams replaced SAR1 rams (14 April), which were replaced by SAR3 rams (22 May) until 30 June. CR-treated ewe lambs were housed with the unstimulated rams. Ovulation was identified by weekly plasma progesterone concentrations and estrous behavior, as indicated by colored rumps. A greater proportion of SAR than CR ewe lambs ovulated in April (52% vs. 0%) and May (68% vs. 0%) (P<0.0001), and were in estrus in May (64% vs. 0%) and June (92% vs. 24%) (P<0.0001). The presence of SAR rams reduced (P<0.0001) mean (±SD) age (d) at first ovulation and estrus (235±28 [7 May] and 257±24 [29 May], respectively), compared to that of CR-treated ewe lambs (277±5 [18 June] and 302±16 [14 July], respectively). In conclusion, the presence of photoperiod-melatonin-induced, sexually activated rams in spring, advanced puberty in autumn-born ewe lambs. Using this technique might provide an effective and sustainable means of increasing the productive life of ewes, while avoiding the use of hormonal treatments.

Continuous exposure to sexually active rams extends estrous activity in ewes in spring.

Sexual activity in sheep is under photoperiodic control, which is the main environmental factor responsible for the seasonality of reproduction. However, other natural environmental factors such as presence of conspecifics can slightly influence the timing of onset and offset of the breeding season. In goats, we have found that the continuous presence of bucks that were rendered sexually active out of season by previous exposure to long days, prevented goats from displaying seasonal anestrus, which suggests that the relative contribution of photoperiod in controlling seasonal anestrus should be reevaluated in small ruminant species. The aim of this study was to assess whether the presence of sexually active rams that had been stimulated by artificial photoperiod and melatonin implants, reduces seasonal anestrus in sheep, by prolonging ovulatory activity in spring. Ewes were assigned to one of two groups (n = 16 and 15), which were housed in two separate barns, and kept in contact, either with the treated or the control rams between March and July. Vasectomized rams were either exposed to 2 months of long days followed by the insertion of three subcutaneous melatonin implants (treated rams, n = 8), or exposed to natural light conditions (control rams, n = 2). Estrus was monitored daily, and weekly plasma progesterone analyses indicated ovulatory activity. Ewes that were exposed to treated rams exhibited a higher proportion of monthly estrus than ewes exposed to the control rams (P < 0.05). Thirteen of 15 ewes (one ewe was not considered because of the presence of persistent CL) exposed to stimulated rams exhibited estrous behavior in a cyclic manner. In contrast, all ewes exposed to control rams stopped estrous activity for a period of time during the study, such that this group exhibited a significantly longer anestrous season (mean ± standard error of the mean 89 ± 9 days) than did the ewes housed with treated rams (26 ± 10 days; P < 0.0001). Among 15 ewes housed with treated rams, 13 of them exhibited continuous ovulatory activity between March and July, whereas one stopped in June and two in July. All ewes kept with control rams stopped ovulating for some time; consequently, those ewes had a longer anovulation period than did the group exposed to treated rams (3 ± 3 vs. 18 ± 7 days, respectively; P < 0.05). In conclusion, continuous exposure to sexually activated rams induced by artificial photoperiod and melatonin implants in spring extended the ovarian activity of ewes in spring, which results in an increase in estrous expression.

Undernutrition affects embryo quality of superovulated ewes.

To determine the effect of undernutrition on embryo production and quality in superovulated sheep, 45 ewes were allocated into two groups to be fed diets that provided 1.5 (control, C; n = 20) or 0.5 (low nutrition, L; n = 25) times daily requirements for maintenance, from oestrous synchronization with intravaginal sponges to embryo collection. Embryos were collected 7 days after the onset of oestrus (day 0). Low nutrition resulted in lower live weight and body condition at embryo collection (P < 0.05). Diet (P < 0.01) and day of sampling (P < 0.001) significantly affected plasma non-esterified fatty acid (NEFA) and insulin concentrations. Plasma leptin concentrations decreased on day 7 only in L ewes. A significant effect of dietary treatment (P < 0.05) and day (P < 0.0001) was observed on plasma insulin-like growth factor (IGF)-I concentrations. The number of recovered oocytes and embryos did not differ between the groups (L: 15.4 ± 0.4; C: 12.4 ± 0.4). Recovery rate was lower (P < 0.05) in the L (60%) than in the C group (73%). The total number of embryos and number of viable-transferable embryos (5.0 ± 0.3 and 3.4 ± 0.3 embryos, respectively) of the L group were lower (P < 0.1) when compared with controls (8.4 ± 0.4 and 6.2 ± 0.4 embryos, respectively). Undernutrition during the period of superovulation and early embryonic development reduced total and viable number of embryos. These effects might be mediated by disruption of endocrine homeostasis, oviduct environment and/or oocyte quality.

Periconceptional undernutrition modifies endocrine profiles and hepatic gene expression in sheep.

This study investigated whether a 22-day period of undernutrition (half maintenance) could affect maternal endocrine responses and liver gene expression during early pregnancy (day 7). Thirty-five ewes were fed 1.5 (n = 15) or 0.5 (n = 20) their maintenance requirements and slaughtered on day 7 of the oestrus cycle or pregnancy (oestrus = day 0). Insulin, IGF, leptin and non-esterified fatty acids (NEFA) were determined on days -14, 0 and 7. Transcripts of the IGF family and adipokines receptors were determined in the liver by real-time RT-PCR. Underfed animals presented lower body weight and body condition, greater plasma concentration of NEFA, and lower plasma concentrations of leptin, insulin and IGF1 compared to adequately fed animals. Underfed ewes presented greater hepatic expression of IGFBP2 than well-fed ewes, but tended to have lesser expression of IGFBP5. While no effect of undernutrition on IGFBP4 and ADIPOR2 mRNA expressions was observed, they were increased by pregnancy in underfed animals. This study shows that undernutrition modifies endocrine profiles and hepatic gene expression of IGFBP2 and 5. The pregnancy status increased hepatic gene expression of IGFBP4 and ADIPOR2 mRNA in undernourished ewes.

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.

The effect of periconceptional undernutrition of sheep on the cognitive/emotional response and oocyte quality of offspring at 30 days of age.

Maternal periconceptional undernutrition is associated with altered development and increased risks of adverse outcomes in the offspring. This circumstance is normal in flocks under extensive farming systems, which depend on natural forage resources. The aim of this study was to determine the effect of periconceptional undernutrition in sheep on behavioral and reproductive aspects of the offspring. Eighty ewes were synchronized in estrus and allocated to two groups (n=40) to be fed diets that provided 1.5 (C) or 0.5 (L) times the requirements for maintenance. Ewes were mated and 7 days later fed the control diet until lambing. One month after lambing, 32 lambs were exposed to tests to determine their cognitive and emotional responses. Six ewe lambs were euthanized and in vitro maturation and fertilization procedures were performed. L ewes presented a significant reduction in prolificacy and fecundity (P<0.05) in comparison with C ewes. Mean LW at lambing of L lambs was significantly higher than C lambs (C: 3.80 ± 0.11; L: 4.24 ± 0.15 kg, P<0.05). Lambs born from C ewes spent more time walking than L lambs (P<0.05) in the isolation test, revealing a decrease in the locomotor activity of lambs born from undernourished ewes around conception. Ewe lambs from the undernourished ewes presented a total population of oocytes 2.3 times higher than ovaries from control ewe lambs (60.0 ± 7.8 v. 140.0 ± 18.5 oocytes; P<0.05). In conclusion, periconceptional undernutrition is able to produce an increment in the body weight and the oocyte population, and an alteration of the locomotor activity of the offspring.

Effect of exogenous melatonin on embryo viability and uterine environment in undernourished ewes.

The effect of exogenous melatonin on embryo viability in undernourished ewes was investigated. At lambing, 24 ewes were treated (+MEL) or not (-MEL) with a melatonin implant. After 45 days, both groups were fed to provide 1.5 (Control, C) or 0.5 (Low, L) times daily maintenance requirements, so that experimental groups were: C-MEL, C+MEL, L-MEL and L+MEL. Ewes were mated (Day 0) and on Day 5 embryos were recovered and classified according to their developmental stage and morphology. Ovaries were used for in vitro fertilization and uterine horns were processed to study progesterone and oestrogen receptor (PR and ERα) expression by inmunohistochemistry. After 21 days, groups L-MEL and L+MEL had an average weight loss of 10kg (P<0.001). Number of viable embryos per CL from L+MEL (0.50±0.2) was higher than from other groups (P<0.05). Overall, the melatonin effect was particularly evident in undernourished ewes, increasing both viability (L+MEL: 65%; L-MEL: 25%; P<0.05) and pregnancy rates (L+MEL: 66.6%; L-MEL: 16.6%; P<0.05). Neither nutrition and melatonin nor their interaction had a significant effect on the in vitro oocyte development. Melatonin treatment tended to increase the percentage of positive cells to PR in deep glandular epithelium, independently of diet (P=0.09), and the greatest staining intensity of PR was observed in the luminal and superficial glandular epithelia (P<0.0001). In conclusion, melatonin implants at lambing during the breeding season improve the viability of embryos recovered from undernourished ewes, although this effect seems not to be mediated at the oocyte competence level.

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.

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.

Effects of exogenous melatonin on in vivo embryo viability and oocyte competence of undernourished ewes after weaning during the seasonal anestrus.

This study investigated the effects of exogenous melatonin on embryo viability and oocyte competence in post-partum undernourished ewes during the seasonal anestrus. At parturition (mid-Feb), 36 adult Rasa Aragonesa ewes were assigned to one of two groups: treated (+MEL) or not treated (-MEL) with a subcutaneous implant of melatonin (Melovine(R), CEVA) on the day of lambing. After 45 d of suckling, lambs were weaned, ewes were synchronized using intravaginal pessaries, and fed to provide 1.5x (Control, C) or 0.5x (Low, L) times daily maintenance requirements. Thus, ewes were divided into four groups: C-MEL, C+MEL, L-MEL, and L+MEL. At estrus (Day=0), ewes were mated. At Day 5 after estrus, embryos were recovered by mid-ventral laparotomy and classified based on their developmental stage and morphology. After embryo collection, ovaries were recovered and oocytes were classified and selected for use in in vitro fertilization (IVF). Neither diet nor melatonin treatment had a significant effect on ovulation rate and on the number of ova recovered per ewe. Melatonin treatment significantly improved the number of fertilized embryos/corpus luteum (CL) (-MEL: 0.35 +/- 0.1, +MEL: 0.62 +/- 0.1; P = 0.08), number of viable embryos/CL (-MEL: 0.23 +/- 0.1, +MEL: 0.62 +/- 0.1; P < 0.01), viability rate (-MEL: 46.6%, +MEL: 83.9%; P < 0.05), and pregnancy rate (-MEL: 26.3%, +MEL: 76.5%; P < 0.05). In particular, exogenous melatonin improved embryo viability in undernourished ewes (L-MEL: 40%, L+MEL: 100%, P < 0.01). Neither nutrition nor exogenous melatonin treatments significantly influenced the competence of oocytes during IVF. Treatment groups did not differ significantly in the number of healthy oocytes used for IVF, number of cleaved embryos, or number of blastocysts and, consequently, the groups had similar cleavage and blastocyst rates. In conclusion, melatonin treatments improved ovine embryo viability during anestrus, particularly in undernourished post-partum ewes, although the effects of melatonin did not appear to be mediated at the oocyte competence level.

Short-term undernutrition affects final development of ovulatory follicles in sheep synchronized for ovulation.

The objective of this study was to determine, in sheep, the effect of a short-term undernutrition on growth dynamics and competence of pre-ovulatory follicles. Synchronization of sexual cycles and induction of ovulation were performed, with progestagens and gonadotrophins, in 14 adult female sheep. Morphological characteristics and developmental competence of ovarian follicles to achieve ovulation were determined by imaging techniques (ultrasonography and laparoscopy) and blood sampling. All the animals ovulated and mean ovulation rates were similar between groups (2.0 ± 0.6 corpora lutea in control ewes and 2.2 ± 0.8 in undernourished sheep). However, nutritional restriction, even during a short period, was related to the presence of large follicles in static growing phase which, despite reaching ovulation, persisted static during the induced follicular phase and evidenced functional alterations as there was no inhibition of the development of subordinate follicles. Thus, this study suggests the existence of deleterious effects from short-term undernutrition on functionality of pre-ovulatory follicles, which can compromise fertility.

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 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.

Effect of undernutrition on the uterine environment during maternal recognition of pregnancy in sheep.

The effects of pregnancy and undernutrition on endometrial gene expression were investigated in ewes fed all or half their maintenance requirements and killed on Day 14 of pregnancy or of the oestrous cycle. The endometrial expression of progesterone, oestrogen, oxytocin and interferon receptors (PR, ERalpha, OXTR and IFNAR, respectively), cyclo-oxygenase (COX)-2, proliferating cell nuclear antigen (PCNA), insulin-like growth factors (IGF)-I and -II, and IGF-1 receptor (IGF-1R) was studied by immunohistochemistry or real-time reverse transcription-polymerase chain reaction. The luminal epithelium of cyclic control ewes was devoid of PR staining and had relatively high levels of ERalpha, OXTR, COX-2 and IFNAR2. The presence of a conceptus decreased the in vitro uterine secretion of prostaglandin (PG) F(2alpha) and the expression of IFNAR2 in most cell types, and increased the gene expression of IGF-I and IGF-II. Undernutrition tended to increase ERalpha protein and gene, but decreased in vitro uterine secretion of PGE(2) and the gene expression of IFNAR2 in cyclic ewes. There was no effect of undernutrition on pregnancy rates or the number of conceptuses recovered. Consistent with this, undernutrition of pregnant ewes did not have any effect on uterine gene expression. Moreover, in cases where changes were observed in cyclic ewes, these changes were negated when a conceptus was present.

Effects of annual rainfall and farm on lamb production after treatment with melatonin implants in Merino sheep: a 4-year study.

AIM: To determine the effects of annual rainfall and farm on the efficacy of melatonin implants in improving lamb production in Merino sheep in Spain. METHODS: A study was conducted on 3,871 Merino sheep on six farms over a 4-year period (2004-2007). Melatonin implants were inserted during the second half of February or early March (winter) (Melatonin group) or not (Control group). Multinomial logistic regression was used to determine the effects of melatonin, farm and year, and their interactions, on reproductive outcomes. Regression analysis was used to examine the relationship between annual rainfall and the percentage of ewes lambing, percentage of lambs born to ewes lambing, and overall lambing percentage, for each year and treatment group within farm. RESULTS: Annual rainfall, farm and treatment with melatonin, and their interactions, had a significant effect on the reproductive performance of ewes (p<0.001). Treatment with melatonin increased the percentage of ewes lambing (Melatonin group = 77 (SEM 4)%, Control group = 44 (SEM 7)%; p<0.0001), and overall lambing percentage (Melatonin group = 109 (SEM 1)%, Control group = 59 (SEM 2)%; p<0.0001). Treatment differences were especially pronounced in 2005 and 2006, when annual rainfall was exceptionally low; ewes in the Control group had the lowest lambing rates those years. Lambing rates and overall lambing percentage were positively correlated (p<0.05) with the amount of annual rainfall but the correlation coefficients were higher in the Control than Melatonin group. CONCLUSIONS: Melatonin implants are an effective means of improving lamb production of Merino ewes, especially in harsh environments where low annual rainfall limits the availability of food. When melatonin treatment was used, however, the responses of flocks on individual farms were difficult to predict because within a year, responses did not occur on all farms.

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.

Early pregnancy alters the metabolic responses to restricted nutrition in sheep.

This study investigated whether a 27-day period of nutrition at half-maintenance during early pregnancy (up to Day 14) could alter maternal endocrine responses. Forty-six ewes were fed all or half of their maintenance requirements and slaughtered on Day 14 of the oestrous cycle or pregnancy. We used real time RT-PCR to study gene expression of growth hormone receptor (GHR) and leptin in adipose tissue and GHR, GHR1A and of the insulin-like growth factor I (IGF-I) in the liver. Blood profiles of metabolites and metabolic hormones were also determined. Throughout the experiment, underfed animals presented lower body weight and body condition, greater plasma concentrations of non-esterified fatty acids (NEFA), and lower plasma concentrations of leptin, compared to adequately fed animals. Undernutrition affected the patterns of gene expression in adipose and hepatic tissues, and the responses differed between pregnant and non-pregnant ewes. In adequately fed ewes, pregnancy up-regulated leptin mRNA expression in adipose tissue, a response that was impaired in underfed ewes. The hepatic expression of IGF-I mRNA was increased by pregnancy in underfed animals while no effect was observed in adequately fed ewes. It remains to be determined whether the changes in the endocrine milieu are paralleled by modifications in uterine gene expression that could alter the environment of the embryo during early pregnancy.

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.

Effect of exogenous melatonin on the ovary, the embryo and the establishment of pregnancy in sheep.

Administration of melatonin to advance the breeding season in sheep has been widely used, since this hormone conveys the photoperiodic signal to the reproductive neuroendocrine axis. An increased lambing percentage has been reported following such treatment during anoestrus, which could be mediated through a higher rate of embryonic survival, either by an improvement in luteal function or by a reduction in the antiluteolytic mechanisms. The aim of this article is to review the body of knowledge on the effect of melatonin on the ovine ovary, the embryo and the establishment of pregnancy. Some studies using synchronized ewes have found that melatonin treatments during anoestrus do not improve ovulation rate by modifying the timing of follicle emergence, but increasing the number of ovulatory follicles by decreasing the atresia of medium and large follicles. On the other hand, the addition of melatonin to the in vitro maturation medium does not improve oocyte maturation rate in oocytes from sheep ovaries recovered either in anoestrus or in the breeding season. However, a luteotrophic effect of melatonin at either short or medium term has been reported. We have recently observed that melatonin implants tend to improve the survival of embryos collected from ewes after superovulation in anoestrus. More specifically, melatonin induced a significant reduction of the number and rate of non-viable (degenerate and retarded) embryos. Preliminary data from our laboratory suggest that the uterine sensitivity to progesterone - in terms of progesterone receptor expression - of superovulated ewes could be reduced by melatonin treatment. It can be concluded that the success of exogenous melatonin as a means to improve lamb production of sheep is due, at least in part, to an improvement of luteal support and embryonic survival.