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Management of the ovine oestrous cycle is mainly based on the use of exogenous hormones to mimic or enhance (progesterone and its analogues) or manipulate (prostaglandin F2α and its analogues) the activity of the corpus luteum, combined with the application of other hormones mimicking the pituitary secretion of gonadotrophins (e.g. equine chorionic gonadotrophin). These protocols have been applied without major change for decades but, now, there are two reasons to reconsider them: (1) our greatly improved knowledge of the dynamics of ovarian physiology, following the application of transrectal ultrasonography, indicates that modification of the protocols may improve fertility yields and (2) increasing concerns about animal health and welfare, food safety and the environmental impact of the treatments, as evidenced by public opinion and therefore market forces. Here, we offer an overview of these issues, introduce an updated protocol and suggest ways for future improvements to the protocols.

The present study supports that short-term (7-days) protocols based on progestagen-impregnated sponges and progesterone-loaded CIDRs are equally effective to induce ovulatory response in sheep. There were no significant differences in the onset of estrus behavior (32.0 ± 6.0 and 33.8 ± 4.0 hr after device withdrawal for sponges and CIDRs, respectively; p > 0.05) and preovulatory LH discharge (5.1 ± 2.1 and 5.8 ± 3.3 hr after onset of estrus behavior for sponges and CIDRs, respectively; p > 0.05). These features are similar to previously described for classical long-term (12-14 days) treatments. Hence, short-term CIDR-based protocols may be implemented using the same time-intervals for insemination than sponge-based and long-term protocols., (© 2018 Blackwell Verlag GmbH.)

This study assessed milk productivity, demographic characteristics and workload distribution on a single high-yield dairy ewe farm in Spain (Avila, Spain; continental climate, latitude of 40.90 N, altitude of 900 m) over a 7-year period considering a transition from a herd management system involving five lambings per year (5LY) to a system involving 10 lambings per year (10LY). The 5LY system was practiced on the farm from 2010 to 2012 and the 10LY system from 2014 to 2015, with 2009 and 2013 being considered transition years. During this period, 27 415 lactations were recorded from an average of 3746 Lacaune sheep/year. Several productivity parameters were higher in 2014 to 2015 than in 2010 to 2012: milk yield/lactation (370±156 v. 349±185 l), lactation length (218±75 v. 192±75 days) and dry period length (53.5±38.3 v. 69.1±34.8 days) (all P<0.0001). During 2014 to 2015, investment in new lambing facilities was possible, workload was distributed more uniformly throughout the year, workload per worker was smaller, rate of ewe culling was lower (35.39±0.53% v. 42.51±7.51%), ewe longevity was greater and higher-order lactations were more numerous (P<0.0001). On the other hand, during 2010 to 2012, daily production was higher (1.73±1.66 v. 1.70±0.62 l/day; P=0.038), the interlambing period was shorter (283±50 v. 302±44 days; P<0.0001) and lambings/ewe per year were greater (1.42±0.01 v. 1.30±0.01; P<0.05). These results suggest that a 10LY herd management system can be compatible with profitability, productivity and good animal and worker's welfare on a high-yield dairy farm, and may even be associated with better outcomes than a 5LY system.

In the present study, there was a comparison among classical long-term progestagen (fluorogestone acetate) protocols for synchronization of estrus and ovulation (14 days; group FGA14, n = 9 ewes) and short-term protocols based on 7 days of progestagen treatment plus a dose of prostaglandin F 2α at either insertion (PG-FGA7, n = 11) or removal (FGA7-PG, n = 12). There were no significant differences in the ovulation rate and progesterone secretion among treatments. The FGA7-PG group, however, had a similar percentage of ewes expressing estrous behavior than the group FGA14 (90.9 and 100%, respectively, with a trend for a lesser percentage in the PG-FGA7 group, 63.6%) and about 90% of the ewes in the FGA7-PG group had the preovulatory surge release of LH 8 h after the onset of estrous behavior. These features may be related to a greater number of preovulatory follicles during growing phases (P < 0.05) and a greater plasma estradiol concentration (P < 0.05) in this group than in the classical 14-day group, which suggest these are more functional preovulatory follicles. In conclusion, therefore, the use of the FGA7-PG treatment may favor efficiency of progestagen-based protocols for reproductive management., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this Research Communication we assessed factors affecting colostrum quality of dairy Lacaune ewes using the Brix-refractometer. Colostrum from 536 lambings from one commercial intensive dairy Lacaune farm were analysed for the following factors with potential influence in colostrum quality: (1) ewe parity (n = 84-132), (2) length of previous dry period (PDP) (n = 23-214), (3) age at first lambing (AFL) of primiparous ewes (n = 9-88), (4) lambing season (n = 192 or 344), and (5) year (2011-2013, n = 142-203). Parity significantly affected colostrum quality, with primiparous ewes showing the highest Brix refractometer values (22·6 ± 5·6%, P < 0·0001), though values were similar among multiparous ewes. PDP length also significantly affected colostrum quality: ewes with the shortest PDP showed the worst quality (16·8 ± 4·2%, P < 0·0001), with quality gradually rising with PDP length. Colostrum quality was significantly higher in 2011 (21·0 ± 5·2%) than in 2012 or 2013 (P < 0·0001); this likely reflects the several-fold greater proportion of animals with long PDP in 2011. In contrast, neither AFL nor lambing season significantly affected colostrum quality. These results suggest that parity and PDP length can substantially affect ovine colostrum quality of dairy ewes under intensive management conditions and they further show the usefulness of the Brix refractometer for providing a rough estimation of colostrum quality on-farm. However, further studies are needed to determine a validated cut-off Brix value for identifying good-quality colostra in ovine species.

Often the only way to ensure profitability of Lacaune dairy sheep is intensive management, which requires appropriate dry-period treatment to ensure animal productivity and health. The present study aimed to investigate the effects of intramammary antibiotic dry therapy on the performance and health of Lacaune sheep under intensive management. We recorded data for 5981 complete lactation periods that followed a dry period. A total of 2402 lactation periods were preceded by a dry period involving intramammary administration of 300 mg of cephapirin benzathine (antibiotic group) and 3579 lactation periods were preceded by dry periods with no treatment (control group). The following on-farm yield data were collected for individual lactation periods: length of the subsequent lactation period; total milk yield per lactation period; daily milk yield and length of the subsequent dry period. Data on confounding factors that might affect productivity were also recorded, including the individual ewe, number of lactation periods and length of the previous dry period. Milk quality was assessed using data on somatic cell count (SCC) and content of protein and fat taken from the Spanish National Official Milk Yield Recording System. Antibiotic dry therapy significantly improved total yield per lactation period, which was 429±151·1 l in the antibiotic group and 412±165·5 l in the control group, as well as the daily milk yield, which was 1986±497·0 and 1851±543·2 ml/d, respectively (both P<0·0001). The initial dry period was significantly longer in the antibiotic group than in the control group, and dry period length correlated inversely with yield variables such us total yield per lactation period (r=-0·055; P<0·0001) and yield per day in milk (r=-0·039; P<0·0001). As a result, milk yield records systematically underestimated the positive effects of antibiotic dry therapy. Antibiotic dry therapy also significantly improved milk quality. Milk from the antibiotic group showed 50% lower SCC (573±1326 vs. 1022±2126 cells/ml; P<0·0001) and slightly higher content in fat (7·33±0·91 vs. 7·15±0·87%) and protein (5·63±0·44 vs. 5·44±0·4%). The results of this study suggest that cephalosporin dry therapy of Lacaune dairy sheep increases milk production and improves milk quality during subsequent lactation periods.

At high altitude, hypoxia and/or oxidative stress may compromise fertility. This study tested the relative effect of short- or long-term exposure to high-altitude hypobaric hypoxia and oxidative stress in sheep on preovulatory follicle dynamics and gonadotrophin secretion. Thus, growth dynamics, stereidogenic function and competence to ovulate of preovulatory follicles, as well as FSH and LH availability throughout the entire oestrous cycle, were compared among sheep native from low and high altitude, and sheep newcomers to high altitude. The results indicates that short-term exposure in sheep newcomers to high altitude has a deleterious effect on both the ovarian function (affecting preovulatory follicular development) and the pituitary function (diminishing plasma LH availability). On the other hand, there were no detected differences in the preovulatory follicular development in sheep adapted to high altitude for generations and, conversely, LH secretion was increased, which suggests an adaptive mechanism. The treatment with antioxidant agents during a relative short period for the time of folliculogenesis (approximately 1 month and a half) changed substantially the development of preovulatory follicles in short-term exposed sheep to similar patterns than in sheep native and living to both high and low altitude. These results highlight the role of oxidative stress in the detriment of the reproductive function in individuals recently exposed to high-altitude hypoxic environment., (© 2014 Blackwell Verlag GmbH.)

Reproductive outputs in fat-tailed Barbarine sheep in central Tunisia are often low because of feed shortage and the low nutritive value of diets. Supplementation with conventional concentrates is economically unsuitable in central Tunisia, so more cost-effective and sustainable alternative feeding strategies need to be developed. We tested effects of short-term nutritional treatments including cactus cladodes during the induction of 'male effect' on fertility and prolificacy parameters (follicular growth, ovulatory response and early embryo losses). One hundred and twenty ewes were distributed in 4 equal groups balanced for live weight grazed natural pastures and were supplemented for 21 days, starting day 10 after introduction of rams, with cactus cladodes (CA), cactus cladodes and soybean meal (CAS), concentrate (CC) or only soybean meal (S). Nutritional treatment did not affect live weight in this experiment. Ewes receiving cactus had higher number of large pre-ovulatory follicles (≥6 mm; 1.08 ± 0.05), between days 14 and 19 after introduction of rams, than females in the CC and S ewes (0.64 ± 0.06; p < 0.05). However, there were no differences in the onset of oestrous behaviour in response to 'male effect' or in the number of corpora lutea. Average ovulation rates were 1.42 ± 0.16 for CC, 1.47 ± 0.13 for CAS, 1.31 ± 0.15 for CA and 1.31 ± 0.13 for S groups respectively. Finally, reproductive wastages at day 35 after mating were not different between groups being 0.33 ± 0.19 for CC, 0.60 ± 0.17 for CAS, 0.43 ± 0.16 for CA and 0.31 ± 0.15 for S groups respectively. It is concluded that Barbarine ewes fed nutritional treatments including cactus performed similarly to those receiving diets including conventional concentrate feeds., (© 2013 Blackwell Verlag GmbH.)

The present study evaluated the effects of supplementation with cactus cladodes on follicular dynamics and ovulatory response of sheep reared in semi-arid areas. A total of 76 ewes were distributed into two equal groups supplemented with either concentrated feed or cactus cladodes. After 30 days of supplementation, no differences were found between feeding regimens on the final live weight (LW; 41.5 ± 0.6 and 42.1 ± 0.7 kg in the Concentrate and Cactus groups respectively) and body condition score (BCS; 1.8 ± 0.3 and 1.8 ± 0.4 for Concentrate and Cactus group respectively). Moreover, no differences were found between the initial and the final values of both LW and BCS; thus, there were no effects of supplementation on any of both parameters. Analysis of follicular population showed that, during the follicular phase induced by ram effect, the number of follicles reaching ovulatory size increased in both groups. However, the number was always higher in Cactus ewes and, at oestrus, Cactus ewes had 1.6 ± 0.2 and Concentrate sheep had 1.2 ± 0.2 large follicles (p < 0.05). Thereafter, ovulation rate was affected by duration of supplementation; being higher in sheep fed with cactus for 6-10 days (1.7 ± 0.1) than in ewes supplied with cactus for more than 11 days (1.3 ± 0.1; p < 0.05), in sheep fed with concentrate for 6-10 days (1.2 ± 0.1; p < 0.01) and even than in individuals subjected to classical flushing with concentrate (1.3 ± 0.1; p < 0.05)., (© 2011 Blackwell Verlag GmbH.)

The present study aimed to determine the influence of a glucogenic supply on oocyte developmental competence. Oestrous cycles were synchronised in 22 Sarda ewes by the insertion (Day 0) of one intravaginal progestagen-impregnated sponge that was removed after 6 days. After removal, the ewes were randomly allocated into two experimental groups (treated and control ewes) and, from Day 7 to Day 11, treated ewes received oral administration of a glucogenic mixture, whereas control animals received water. Follicular development was stimulated by FSH administration from Days 8 to 10. Glucose metabolism was assessed from Days 7 to 11, whilst follicle and corpus luteum growth dynamics and functionality were evaluated between Days 6 and 11. At Day 11 ovaries were collected and processed for in vitro embryo production. Glucogenic treatment increased both the plasma levels of glucose, progesterone, oestradiol and the number of 2-3-mm follicles (P < 0.05). Higher fertilisation and blastocyst rates (P < 0.05) were obtained after IVM of oocytes recovered from treated ewes compared with control ones. In conclusion, glucogenic treatment modifies follicle and corpus luteum functionality and improves oocyte quality, as evaluated by in vitro developmental kinetics and blastocyst output.

The aim of the current study was to determine the possible effects of progestagen oestrous synchronization on vascular endothelial growth factor (VEGF) expression during sheep luteogenesis and the peri-implantation period and the relationship with luteal function. At days 9, 11, 13, 15, 17 and 21 of pregnancy, the ovaries from 30 progestagen treated and 30 ewes cycling after cloprostenol injection were evaluated by ultrasonography and, thereafter, collected and processed for immunohistochemical evaluation of VEGF; blood samples were drawn for evaluating plasma progesterone. The progestagen-treated group showed smaller corpora lutea than cloprostenol-treated and lower progesterone secretion. The expression of VEGF in the luteal cells increased with time in the cloprostenol group, but not in the progestagen-treated group, which even showed a decrease between days 11 and 13. In progestagen-treated sheep, VEGF expression in granulosa-derived parenchymal lobule capillaries was correlated with the size of the luteal tissue, larger corpora lutea had higher expression, and tended to have a higher progesterone secretion. In conclusion, the current study indicates the existence of deleterious effects from exogenous progestagen treatments on progesterone secretion from induced corpora lutea, which correlate with alterations in the expression of VEGF in the luteal tissue and, this, presumably in the processes of neoangiogenesis and luteogenesis., (© 2010 Blackwell Verlag GmbH.)

The aim of the current study was to determine possible differences in ovarian and pituitary features explaining lower fertility rates in sheep with oestrus induced with intravaginal progestagens or prostaglandin analogues (group FGA and PGF, n=8 in both) when compared to a control group (group C, n=8). The growth profiles and the mean individual sizes of preovulatory follicles were similar between groups; however, the number of preovulatory follicles per ewe and, consequently, the number of ovulations were higher in groups FGA and PGF (2.3±0.3 and 2.0±0.1, respectively) than in group C (1.4±0.1, P<0.05). However, plasma oestradiol concentrations were similar between groups suggesting a defective function in some preovulatory follicles of groups FGA and PGF. In group FGA, the basal LH levels during the follicular phase were lower (0.21±0.0 ng/mL, P<0.005) than in groups C (0.41±0.1 ng/mL) and PGF (0.55±0.1 ng/mL); the onset of preovulatory discharge being later (21.0±2.3h vs. 12.8±1.5 in C and 14.5±1.5 in PGF; P<0.05 for both). Finally, luteal activity was also found to be affected in group FGA; the rate of progesterone secretion per total luteal tissue was lower (range: 0.46-0.65 ng/mL/cm(2)) than in ewes treated with cloprostenol (2.1-3.3 ng/mL/cm(2)) and control sheep (2.0-3.4 ng/mL/cm(2))., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The present study investigated the effect of age at first lambing (AFL) on the performance of Lacaune sheep under intensive management conditions. Records from 3088 maiden sheep from one farm, for the period 2005-2010, were classified into four experimental groups: group E (early) ewes with AFL ⩽390 d; group M (middle) with AFL of 391-450 d; group L (late) with AFL of 451-510 d; and group A (aged) with AFL ⩾511 d. The higher the number of lactations, the lower were the yield/lactation and yield/Day in Milk. Ewes from group M up to 450 days old lambed 0.2 times more often and had 0.25 more lactations than the ewes from group L; in addition, the former group lambed 0.5 times more often and had 0.49 more lactations than ewes from group A. Group A had the lowest yield per lifetime; the yield followed the sequence: group E (1032 l) >group M (1051 l) >group L (989 l) > group A (859 l) (P<0.0001). Yield/lactation was affected by AFL during the first three lactations (P<0.05). Group E produced significantly less milk in the first lactation than ewes from groups M, L and A. AFL correlated negatively with the number of lactations in life (r= -0.26; P<0.0001) and with total milk yield (r= -209; P<0.0001). The productive performance between the ewes that reached puberty early (n=404) or late (n=2684) in life were different (P<0.0001) with the early sheep having a lower total milk yield (591 v. 1073 l, P<0.0001). In conclusion, the current study indicates that the optimal AFL for the Lacaune breed under an intensive dairy system is between 390 and 450 d. Ewes with AFL earlier than 390 d or later than 450 d are likely to have a shorter productive life and a lower lifetime milk production.

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., (© 2009 Blackwell Verlag GmbH.)

The effects of supplemented protein level (PL) during the periconceptional period and their interaction with body condition were evaluated in sheep. Multiparous Rambouillet ewes (n = 12) received two PL of rumen undegradable protein (UIP) during a 30-day pre-mating and 15-day post-mating period: low [LPL, 24% crude protein (CP), 14 g UIP and 36 g/CP animal/day] and high [HPL, 44% CP, 30 g UIP and 50 g/CP animal/day]. While ovulation rate (OR) did not differ between treatments (1.6 +/- 0.5, mean +/- SEM), a lower fertility rate, a decreased embryo number and a reduced uterine pH (UpH) was observed in the HPL group (p < 0.05), irrespective of BC. Luteal tissue weight, volume and progesterone secretion did not differ among treatments. Sheep with lower UpH also had lower conceptus weight (Cwt; p < 0.05, r = 0.65) and conceptuses with lower mass tended to secrete less INF-tau and IGF-1, and the correspondent endometrial explants had a higher basal PGF(2alpha) release. Current study indicates that high protein diets during the periconceptional period in sheep modify uterine and embryonic relationships, increasing early pregnancy losses and inducing embryo growth retardation. Surviving embryos were affected by weight reductions, which could compromise later foetal growth and birth weight. Results evidence the key role of a balanced diet in reproductive success and indicate that the quality and nutrient composition of the maternal diet are essential for an adequate establishment of pregnancy, having paramount effects on the interplay of the embryo and the uterus.

The plasticizer di(2-ethylhexyl)phthalate (DEHP), used for producing polyvinyl chloride (PVC), acts as an endocrine disruptor with toxic effects on reproductive and developmental processes. Exposure to DEHP in humans is mainly by environment and food. Thus, our aim was to determine plasma levels in livestock animals using the ewe (Ovis aries) as a model. In a first trial, 150 samples from ewes of different ages (2 to 7 yr) and reproductive status (pregnant and nonpregnant) were analyzed by high-performance liquid chromatography (HPLC). DEHP was detected in 34.7% of the samples, with a mean level of 0.45+/-0.01mug/mL (range, 0.05 to 2.81mug/mL). The percentage of nonpregnant animals with DEHP traces was higher in animals older than 4 yr (n=66, 37.9%) than in younger animals (n=69, 17.4%; P<0.05), although the mean levels in ewes with residues were similar (0.16+/-0.01 vs. 0.16+/-0.02mug/mL). All the pregnant ewes (n=15) showed presence of DEHP, with higher plasma levels than that in nonpregnant females (1.42+/-0.18 vs. 0.16+/-0.01mug/mL; P<0.0001). For confirming the effect of pregnancy on mobilization of DEHP from body fat, 101 ewes of the same age were sampled in a second trial at a different farm. The percentage of animals with DEHP traces was higher in pregnant ewes (n=32, 71.9%; P<0.005) than in nonpregnant ewes (n=37, 35.1%) or in ewes that recently gave birth (n=32, 21.9%), although mean levels were similar (0.42+/-0.02, 0.33+/-0.02, and 0.34+/-0.05mug/mL, respectively). In conclusion, current results indicate a high incidence of ewes reared in the field showing accumulation of phthalates; percentage of animals with presence of DEHP increases with age, due to an extended period of exposure, but mainly during pregnancy, due to the mobilization of body reserves.

The present study aimed to assess the efficacy of reduced doses of cloprostenol for synchronizing estrus and ovulation in hair sheep. With the aim to evaluate the luteolytic activity of reduced cloprostenol doses, a first experiment was performed using a relatively large (group H: 126 microg; n=8), medium (group M: 68.25 microg; n=6) and small (group L: 38.5 microg; n=6) cloprostenol dose. Luteolysis was assessed at Days 3 and 6 after injection (Day 0) by progesterone concentrations (P(4)) and transrectal ultrasonography (US). In Experiment 2, sheep were randomly assigned to the same three doses to evaluate a protocol for estrous synchronization using two injections administered 9 days apart. A third trial was performed with ewes treated (9 days apart) with the large dose (H=126 microg; n=12) and with a small dose adjusted for facilitating volume management (LA=43.75 microg; n=12). Presence of estrous cycling was determined in all the ewes by US and P(4) assay, at Days -9, -6, -2, 0 (Day of second cloprostenol injection), 8 and 11. Bleeding and US were done every 4h from 16 h of the beginning of the estrus during the third trial to assess the preovulatory LH surge and timing of ovulation. Additionally, blood samples were drawn at Days 0, 1, 2 and 3 to assess estradiol (Experiments 2 and 3) and P(4) (Experiment 2) concentrations during the ovarian follicular phase. In all experiments, percentage of animals showing luteolysis, preovulatory follicular dynamics and function and percentage of ewes showing behavioral estrus in response to treatment was similar among groups. Timing of estrus for group H was earlier than group L (28.6+/-1.8h compared with 37.1+/-2.4h; P<0.05). In the third trial, the preovulatory LH peak was higher in the LA group than group H, in terms of maximum mean concentration during the surge (27.7+/-1.8 ng/mL compared with 21.3+/-2.2 ng/mL; P<0.05) and area under the curve (AUC; 183.4+/-12.7 ng/mL compared with 127.7+/-10.9 ng/mL; P<0.01). However, timing of ovulation was similar for H and LA groups. Thereafter, ovulation rate and luteal function at Day 11 were similar. Current results demonstrate that reduced doses of cloprostenol may be applied in a practical manner for reproductive management of sheep, with the additional advantage of reducing treatment costs.

This study assessed the efficacy of a protocol combining short-interval cloprostenol-based protocols and "male effect" for estrous synchronization in hair sheep. In Experiment 1, 24 ewes were randomly assigned to three groups (n=8) and treated with cloprostenol on Days 3, 5 and 7 after ovulation, respectively. Estradiol secretion during the follicular phase was similar among groups. Onset of estrus (P<0.001) and the timing of maximum LH concentration (P<0.01) were earlier in group D3 than in D5 and D7 groups. During the subsequent cycle, the number and size of corpora lutea were higher (P<0.05) in ewes of the groups D3 (1.9+/-0.3 and 115.1+/-14.3mm(2)) and D5 (1.8+/-0.2 and 100.2+/-11.2mm(2)) than in group D7 (1.3+/-0.2 and 75.6+/-6.4mm(2)) group. In Experiment 2, 24 ewes were treated with two cloprostenol injections (7 days apart). Twelve ewes were exposed to "male effect" previous to an isolation period (ME group), whereas the remaining ewes were controls without male exposure (CTR group). Male effect induced earlier preovulatory LH surge (P<0.05) and ovulation (P<0.001) than CTR group. In Experiment 3, the estrus was synchronized in 68 ewes. Nineteen of them (group FGA) were treated using intravaginal sponges impregnated with fluorogestone acetate for 12 days and inseminated at 55h. Forty-nine females (group ME) were treated like ME group. Twenty-four (ME48 group) and 25 ewes (ME55 group) were inseminated at 48 and 55h after treatment, respectively. The fertility rate was numerically higher in ME48 than ME55 and FGA groups (62.5, 44.0 and 47.4%, respectively). In conclusions, the combined use of short-interval cloprostenol treatment and "male effect" may be an adequate alternative for synchronizing estrus and applying artificial insemination in hair sheep throughout the entire year.

Although various progestagens are often used to induce and synchronize estrus and ovulation in ruminants, concerns regarding residues are the impetus to develop alternative approaches, including reduced doses of progestagens. Therefore, the objective was to determine whether ovarian function was affected by halving the dose of fluorogestone acetate in intravaginal sponges for synchronizing ovulation in sheep during the physiologic breeding season. Twenty Manchega ewes, 4-6-year-old, were randomly allocated to receive an intravaginal sponge containing either 20mg (P20, n=10) or 40 mg of fluorogestone acetate (P40, n=10). Cloprostenol (125 microg) was given at sponge insertion, and all sponges were removed after 6d. Ovarian follicular dynamics (monitored by daily ultrasonography) and other aspects of ovarian function did not differ significantly between the two groups. Ovulatory follicles (OF) grew at a similar growth rate (r=0.62; P<0.001), with comparable initial and maximum diameters (4.2+/-0.4 to 6.0+/-0.3mm in P20 vs. 4.6+/-0.6 to 5.7+/-0.2 mm in P40, mean+/-S.E.M.). Plasma estradiol concentrations (determined once daily) increased linearly during the 72 h interval after sponge removal (1.3+/-0.1 to 3.3+/-0.1 pg/mL for P20, P<0.005 and 1.4+/-0.1 to 3.1+/-0.2 pg/mL for P40, P<0.005). Ten days after sponge removal, ovulation rates (1.2+/-0.2 for P20 and 1.4+/-0.3 for P40), and plasma progesterone concentrations (3.8+/-0.35 ng/mL for P20 and 3.9+/-0.38 ng/mL for P40) were similar. In conclusion, reducing the dose of fluorogestone acetate from 40 to 20mg did not affect significantly ovarian follicular dynamics or other aspects of ovarian function.

This study aimed to determine the best vehicle and administration route for progesterone administration in sheep. In a first replicate, single intramuscular doses of 25mg progesterone were administered to ewes previously ovariectomized, either in propylene glycol (group IM-PG, n=6) or olive oil (group IM-OO, n=5). In a second replicate, the same solutions of progesterone were administered subcutaneously to the same ewes (groups SC-PG, n=6, and SC-OO, n=5). In the present study, the best pharmacokinetic results of a single dose of 25mg of progesterone were obtained, both using PG and OO as vehicles, by the subcutaneous route. Thus, progesterone remained in plasma for a longer time after subcutaneous administration in PG than in OO (t(1/2beta): 60.65+/-13.07 vs. 27.51+/-3.59 h; P<0.05); the mean residence time being higher in SC-PG than in SC-OO group (88.99+/-18.36 vs. 41.04+/-5.31h; P<0.05). However, both vehicles allowed maintained plasma levels 0.5 ng/ml for at least 30 h, so any of these treatments may be efficiently used for administration of exogenous progesterone.

Objective: To evaluate the individual functionality of gonadotropin-stimulated preovulatory follicles, for understanding embryo failure in assisted reproductive technique cycles, in a sheep model., Design: Observational, model study., Setting: Public research unit., Animal(s): Fifteen adult Manchega ewes., Intervention(s): Synchronization of the estrous cycle with intravaginal progestagens and ovarian stimulation with FSH; evaluation of reproductive activity, plasma sampling, ovarian ultrasonography, and ovariectomies., Main Outcome Measure(s): Determination of estrus behavior, plasma and intrafollicular concentrations of E(2) and inhibin A, number and size of ovarian follicles, and developmental competence of oocytes., Result(s): These results support the usefulness of serial measurements of plasma inhibin A for assessment of follicular growth during the FSH treatment, rather than of E(2) assays commonly used. Functionality of FSH-stimulated preovulatory follicles is clearly disturbed, as confirmed by a negative correlation between follicular size and intrafollicular concentrations of inhibin A and E(2) in preovulatory follicles after individual dissection; moreover, the ability of their oocytes to resume meiosis was diminished., Conclusion(s): Functionality of follicles in controlled ovarian stimulation (COS), and developmental competence of their oocytes, is disturbed by the high doses of gonadotropin supplied and finally determined by follicular sizes at starting FSH treatment.

Objective: To evaluate intraovarian dominance effects in a sheep model and to determine whether local factors, independent of FSH-mediated pathways, are capable of interfering with the final development of subordinate follicles., Design: Observational, model study., Setting: Public research unit., Animal(s): Three hundred sixty-four adult Manchega ewes., Intervention(s): Synchronization of estrous cycle with intravaginal progestogens and ovarian stimulation with FSH; ovarian ultrasonography, laparoscopies, and embryo recoveries., Main Outcome Measure(s): Determination of morphological characteristics and developmental competence of ovarian follicles., Result(s): The presence of a dominant follicle affects number and viability of embryos from the ipsilateral ovary in females that are treated with high FSH doses; this effect, modulated by season and phase of the estrous cycle, highlights the role of LH in follicular dominance., Conclusion(s): The current study confirms, in a sheep model, the existence of local effects from dominant follicles over neighboring follicles, by direct action and independently from systemic pathways through FSH modulation.

The effect of a short-term nutritional supplementation with steam-flaked corn on metabolism and folliculogenesis was evaluated in 14 maiden sheep. Oestrus was synchronized with two prostaglandin F(2alpha) doses given 10 days apart. From day 11 to 15 of the oestrous cycle induced with prostaglandins, half of the ewes (group 2M) were supplemented with steam-flaked corn, double the daily maintenance ration of the control sheep (group 1M). Body weight and condition remained unaffected, but the energetic supply increased plasma concentrations of glucose (3.6 +/- 0.1 vs 4.3 +/- 0.1 mmol/l, p < 0.0001) for the first 4 days and 3-hydroxybutyrate (0.323 +/- 0.58 vs 0.582 +/- 0.04 mmol/l, p < 0.005) from day 2 to 4. The profile of insulin secretion was also affected by the treatment, increasing in group 2M to reach significant differences on days 13 and 14 (p < 0.05). From similar values at the start of the food supply, the treatment induced a higher follicular development in group 2M (1.1 +/- 1.2 vs 7.4 +/- 1.06 total follicles in day 15, p < 0.05), as evidenced by the lineal increase in the number of larger follicles (>4 mm, p < 0.005). Then, the number of follicles >4 mm in size in 2M was around 60% higher on day 16 (7.86 +/- 0.45 vs 4.86 +/- 0.63, p < 0.005). Thereafter, the mean number of corpora lutea per ewe was around 30% higher in group 2M (1.43 +/- 0.2 vs 1.10 +/- 0.1, although differences were not found to be statistically significant). These data suggest that the use of diets containing high starch sources, like the steam-flaked corn, increases folliculogenesis and ovulation rate in sheep and can be applied in short-term feeding practices.

The present study examines the ovulatory activity of wild and domesticated ewes subjected to either a constant photoperiod of long days (16L:8D) or natural changes in daily photoperiod for 16 mo. The aim was to determine whether an endogenous reproductive rhythm controls seasonal reproductive activity in these sheep, and how the photoperiod might affect this. The effects of long-day photoperiods on long-term changes in prolactin and melatonin secretion were also evaluated. The two species showed changes in reproductive activity under the constant photoperiod conditions, suggesting the existence of an endogenous rhythm of reproduction. This rhythm was differently expressed in the two types of ewe (P < 0.05), with the domestic animals exhibiting much greater sensitivity to the effects of long days. A circannual rhythm of plasma prolactin concentration was also seen in both species and under both photoperiod conditions, although in both species the amplitude was always lower in the long-day animals (P < 0.01). The duration of the nocturnal melatonin plasma concentrations reflected the duration of darkness in both species and treatments. The peak melatonin concentration did not differ between seasons either under natural or long-day photoperiods.

The aims of this study were to evaluate the chronology of periovulatory events (oestrus behaviour, LH surge and ovulation) in 16 superovulated Manchega sheep and to determine whether follicular status at start of the FSH supply might affect their occurrence. Mean timing for onset of oestrus behaviour was detected at 28.1 +/- 0.7 h after sponge withdrawal; the preovulatory LH surge and ovulation started at 37.2 +/- 0.7 h and 65.4 +/- 0.7 h after progestagen withdrawal, respectively. The intervals between oestrus, LH surge and ovulation were affected by a high individual variability, which might be the cause for reported decreased efficiency in embryo production. Current results also addressed the role of follicular status at start of the superovulatory treatment on the preovulatory LH surge and the ovulation. The interval LH surge-ovulation was increased in ewes with a growing dominant follicle at starting the FSH treatment (32.3 +/- 0.9 vs 28.6 +/- 0.5 h, p < 0.05). The developmental stage of the largest follicle at starting the superovulatory treatment also affected occurrence of LH surge and ovulation; follicles in growing phase advanced the occurrence of the LH surge and ovulation when compared to decreasing follicles (33.0 +/- 1.0 vs 43.5 +/- 1.1 h, p < 0.05, for LH peak and 60.7 +/- 1.1 vs 72.8 +/- 1.2 h, p < 0.05, for ovulation). Thus, only ewes with growing follicles ovulated prior to 55 h after sponge withdrawal; conversely, no sheep with decreasing follicles ovulated earlier than 67 h, when an 85.7% of the ewes bearing growing follicles has ovulated at 63 h.

The present study aimed to determine systemic and local effects of corpora lutea (CL), on follicular dynamics throughout the estrous cycle. All follicles >or=2 mm and CL were assessed by daily transrectal ultrasonography in 12 West African ewes. Blood samples were collected to determine plasma concentration of progesterone. Fifteen estrous cycles were evaluated with a mean interovulatory interval of 16.8+/-0.2 days. Two (13.3%), 10 (66.7%) and 3 (20%) of the estrous cycles had 2, 3 and 4 waves of follicular development, respectively. In sheep with three waves of follicular development, both the length of growing phase and the growth rate of dominant follicles from midluteal wave II were diminished (3.4+/-0.3 days, P<0.0001, and 0.4+/-0.1 mm/day, P<0.01, respectively) when compared to follicles from early luteal phase (wave I, 4.1+/-0.2 days, and 0.7+/-0.1 mm/day) or late luteal phase (wave III, 6.3+/-0.4 mm and 0.6+/-0.1 mm/day). The diameter of the dominant follicle was smaller during the midluteal phase (3.9+/-0.1 mm, P<0.0001) than in the early and late luteal phase (5.0+/-0.2 and 5.7+/-0.2 mm; respectively). The effect of the dominant follicle was less during midluteal phase, because number of accompanying smaller follicles was fewer (P<0.01) in waves I and III (6.3+/-0.9 compared with 3.4+/-0.8 and 2.3+/-0.7). The number of follicles was also different between ovaries that had CL and those that did not. The total number of large follicles during the luteal phase was less in ovaries with CL (0.9+/-0.5 compared with 2.7+/-0.3; P<0.01), as was the mean daily number of both large (0.1+/-0.02 compared with 0.2+/-0.02; P<0.001) and total number of follicles >or=2 mm (2.5+/-0.1 compared with 3.3+/-0.1; P<0.01). Current results indicate that the presence of a functional CL may exert both systemic and local effects on the population of follicles, affecting the dominance exerted by large follicles.

In ruminants, nutrition is one of the exogenous inputs affecting reproductive function at different levels of the hypothalamic-hypophyseal-gonadal axis. However, the exact mechanisms or even the identification of the signalling metabolic compounds by which nutrition affects reproductive function still need further clarification. The role of static body condition (BC) and its interaction with a short-term protein supplementation (PL), on secretion of metabolic hormones [growth hormone (GH), insulin and insulin-like growth factor-1 (IGF-1)], as well as on secretion of LH and progesterone (P4) was evaluated in sheep. Twenty-four Rambouillet ewes divided into two groups, with lower (LBC) and higher body condition (HBC), were randomly assigned within BC to one of two PL levels: low (LPL, 24% of crude protein; 14 g/animal/day), and high (HPL, 44% of crude protein; 30 g/animal/day). The secretion of GH, insulin, IGF-1 and LH was evaluated on day 10 of the oestrous cycle; appearance and timing of oestrous behaviour were previously detected using rams. Progesterone secretion was evaluated on day 13 of the same cycle. No differences were found (p > 0.05) between PL groups on serum GH concentrations during the sampling period (overall mean of 4.0 +/- 0.3 ng/ml), but a trend for lower values in HBC sheep was found (3.6 +/- 0.4 vs 4.4 +/- 0.4 ng/ml, p = 0.06). A BC effect was observed (p < 0.05) on serum IGF-1 level, with higher values in HBC sheep (p < 0.05). Neither BC nor PL affected (p > 0.05) secretion of LH and the number of corpora lutea, nor serum P4 and insulin concentrations. Results indicate a predominance of the static component of nutrition on sheep metabolic hormone responses, GH and IGF-1, with no effect of short-term PL on secretion of pituitary and ovarian hormones as well as luteal number and activity.

Possible effects of repeated hormonal treatments and laparoscopic ovum pick-up (LOPU) on the efficiency of oocyte recovery rate and quality were determined in sheep and goats. In six adult Merino sheep and five Criolla goats, ovarian status was synchronized by a prostaglandin F(2 alpha) analogue and the insertion of an intravaginal sponge 48 h later. Follicle development was stimulated by a single dose of FSH (60 mg NIH-FSH-P1) plus a single dose of equine chorionic gonadotrophin (eCG; 300 UI). The first FSH/eCG doses were administered 48 h after the sponge insertion, being repeated every 4 days to complete a total of four treatments in sheep and three in goats. Follicles in both ovaries were categorized according to their diameter and follicular fluid was aspirated under laparoscopic observation without a vacuum pump. In sheep, during a 12-day-period, a total of 347 follicles were aspirated with a recovery rate of 46.9%. In goats, during an 8-day-period, 219 follicles were aspirated with a recovery rate of 45.6%. In both species, there were no significant differences in the number of aspirated follicles, oocyte recovery rate and good quality oocyte recovery rate. However, in sheep the oocyte recovery rate was higher for large follicles, whereas in goats no such effect was detected. In summary, current results indicate that retrieval of oocytes can be maximized, without affecting oocyte quality, by repeating 'oneshot' FSH/eCG regimes and LOPUs at intervals as short as 4 days.

Previous research has reported evidence for negative effects of progestagens on follicular growth and oocyte competence. In the present study, negative effects of progestagens on follicular growth and oocyte developmental competence were assessed. During the breeding season, 20 Sarda ewes were treated with two doses of cloprostenol, 10 days apart, to assure the presence of a corpus luteum (CL). On day 5 after the second cloprostenol dose, 10 ewes were treated with a progestagen sponge while 10 females remained untreated. Starting on day 7 after the second cloprostenol dose, all the ewes were treated with 6 equal doses of 24 I.U. of FSH (Ovagen, ICP, NZ), every 12h. The number of follicles > or =2mm in diameter increased (P<0.0005) in all the ewes from 24 h before to 60 h after the first FSH dose (from 12.8+/-1.1 to 23.4+/-1.3 in treated and from 12+/-0.6 to 22+/-1.2 in untreated ewes, n.s.). There were no significant differences in follicle dynamics between groups, but concentrations of estradiol in control ewes were higher than in the progestagen group (P<0.05). Twelve hours after the last FSH dose, oocytes were collected by ovum pick-up. Recovery rates were lower for progestagen-treated ewes (71.1 versus 83%; P<0.001). After IVP procedure, cleavage rate was also lower in the progestagen group (39.1 versus 82.6%; P<0.001). Furthermore, blastocysts output revealed that oocyte developmental competence was lower in progestagen group (17.3 versus 30.4%; P=0.245), although differences were not significant. These results suggest deleterious effects from progestagen on oocyte developmental competence and set the basis for new protocols for in vitro embryo production.

Current study evaluates effects from breed and management background on follicular dynamics and endocrine output during the follicular phase of sheep oestrous cycle. Follicular phases were synchronized with three cloprostenol doses, 10 days apart, in three groups of 10 females of different non-prolific Spanish breeds (Manchega, Rubia del Molar and Negra de Colmenar). Development of all follicles reaching antral diameters >or=2 mm was assessed by daily transrectal ultrasonographies, whereas follicular function was evaluated by measurement of plasma oestradiol concentrations. All the ovulatory follicles were present at induced luteolysis in Manchega sheep, while a 93.7% were detected in Rubia del Molar and Negra de Colmenar ewes. The mean size of these ovulatory follicles was similar between breeds at 0 h, but their growth rates were higher in Manchega ewes, reaching a larger size at oestrous detection than in Negra de Colmenar and Rubia del Molar sheep (p < 0.05). Conversely, the oestradiol levels increased with time in Rubia del Molar and Negra de Colmenar (p < 0.05); whilst remained stable in Manchega females. However, the patterns of follicular turnover were similar between breeds. These results indicate that, though differences in follicular size and size distribution, patterns of follicular turnover in sheep are affected neither by the breed nor by the background of management and selection.

In the current study we investigated whether the developmental status of the two largest follicles (LF1 and LF2) at the time of administration of the first two doses (0 and 12 h) of FSH of a superovulatory treatment influences periovulatory events and embryo yields in sheep. A larger size of LF1 was negatively correlated with embryo recovery (r=-0.608 for 0 h and r=-523 for 12 h, p<0.05), fertilization (r=-0.464 for 12 h, p<0.05) and viability (r=-0.775 for 12 h, p<0.005). Embryo viability rates were also lower when a higher difference between LF1 and LF2 (r=-0.839 for 0 h and r=-0.761 for 12 h, p<0.01) and a smaller size of LF2 (r=0.877 for 0 h and r=0.622 for 12 h, p<0.01) were observed. This indicates the existence of a limit in the follicular size that will be able to give rise a viable embryo. Conversely, a larger size of LF2 at the time of administration of the first two FSH doses was correlated with reduced recovery rates (r=-0.884 for 0 h and r=-0.706 for 12 h, p<0.01), whilst a decreasing size of LF1 and LF2 was correlated with an increased ovulation rate and recovered embryos. The dominance effect appeared to affect the timing of the preovulatory LH surge. Ewes with a higher difference between LF1 and LF2 displayed earlier LH surges (r=-0.420 for 0 h and r=-0.401 for 12 h, p<0.05) which were related to a higher number of non viable embryos (r=-0.777, p<0.05). The fact that superovulatory yields were affected by, both LF1 and LF2 supports the hypothesis of co-dominance effects in sheep.

Embryo production is a useful tool for ex situ conservation of endangered species and breeds, despite a high variability in the ovarian response to superovulatory treatments. The current study evaluated the incidence and mechanisms of genetic factors in such variability, by determining the pharmacokinetics and pharmacodynamics of a standard treatment with ovine FSH (oFSH) in two endangered Spanish sheep breeds (Rubia del Molar, R, and Negra de Colmenar, N) in comparison to Manchega ewes (M, control group). In the first experiment, pharmacokinetics of an i.m. single dose of 1.32 mg of oFSH was evaluated in seven animals of each breed. Plasma FSH concentrations reached their maximum at 4h post-administration in all the ewes, but several of the kinetic parameters (plasma FSH concentration at 4h post-administration, maximum plasma FSH concentration, C(max), and both the area under the plasma concentration-time curve extrapolated to the infinite, AUC(inf), and to the last moment of sampling, AUC(last)) were higher in the N group. In the second trial, 10 animals of each breed were superovulated using eight decreasing doses of oFSH (3 x 1.32 mg, 2 x 1.10 mg, and 3 x 0.88 mg). The R group, when compared to N and M, showed both a higher number of corpora lutea (13.7+/-0.6 versus 10.0+/-0.4 in N and 9.8+/-0.6 in M, P<0.05 for both) and embryos (7.9+/-0.8 versus 4.3+/-0.4 in N, P<0.05, and 6.7+/-0.5 in M, n.s.). Evaluation of pharmacokinetic and dynamic parameters showed that, although there was a trend for a higher hormone availability in R sheep, mean FSH plasma concentrations were similar between breeds (0.54+/-0.08 ng/ml for R, 0.45+/-0.05 ng/ml for N and 0.35+/-0.05 ng/ml for M). However, differences were found in the number of preovulatory follicles growing in response to the FSH treatment between R (24.4+/-2.2), M (18.9+/-1.5, n.s.) and N sheep (14.1+/-1.4; P<0.01). Thus, differences in embryo yields between breeds would be related to differences in the pattern of follicular growth in response to FSH treatment.

This study evaluated the final output and quality of in vitro produced blastocysts derived from in vivo recovered sheep embryos cultured at various early developmental stages to blastocyst. A total of 270 embryos were recovered from the oviduct, at different days of the early luteal phase, and were classified into three different developmental stages: 2- to 4-cell (n = 93); 5- to 8-cell (n = 92) and 9- to 12-cell (n = 85). The effect of culture conditions was studied, at the same time, by randomly allocating the embryos to one of four groups: three groups of culture with fresh oviduct monolayers (2, 4 and 5 days old) and a fourth group with 2-day monolayers derived from frozen-thawed oviduct cells. Two control groups were established: first, embryos cultured in semi-defined medium (n = 29) and, second, blastocysts obtained in vivo and cryopreserved (n = 43). Influence on blastocyst yield of embryo developmental stage at the start of culture was statistically significant (p < 0.001). Two- to four-cell embryos showed a significantly lower developmental rate (67.7%) than the 5- to 8-cell (83.6%; p < 0.001) and 9- to 12-cell groups (90.5%; p < 0.0001) and lower quality in terms of blastocyst cryotolerance (56.0 vs. 83.7%; p < 0.005). There were no detected effects relating to the age or handling of the monolayer on the embryo developmental rate, but the day of blastocyst appearance was different between embryos cultured on monolayers derived from fresh or frozen-thawed cells (p < 0.0001); the main influence was on the group of 9- to 12-cell embryos (p < 0.0001). Current results confirm the temporal sensitivities of sheep embryos to in vitro culture, regardless of the culture conditions.

This study evaluates the in vitro developmental competence of oocytes collected by ovum pick up (OPU) from sheep treated with GnRH antagonists (GnRHa) and high doses of FSH. Eighteen Sarda ewes were treated with progestagen sponges (day 0). On day 7, 10 ewes received 3 mg of GnRHa s.c., while 8 served as control receiving saline. On day 10, all animals were treated with 96 IU of ovine FSH in four equal doses given i.m. every 12 h. We monitored follicular development by ultrasonography, twice daily from day 7 to 11, and found that GnRHa induced a significant increase in the number of total follicles in 72 h (11.7+/-0.9 to 21+/-2.4, r(2)=0.598, P<0.0001), while this number remained stable in control sheep. We found that FSH induced a significant rise in the number of follicles in both groups; but always higher (P<0.05) in GnRHa treated sheep, confirming that GnRHa enhances ovarian response to exogenous FSH stimulation. Twelve hours after the last FSH dose, oocytes were collected by OPU. Recovery percentage, morphological quality, ability to resume meiosis, fertilization and cleavage were similar in oocytes from treated and untreated sheep. However, the final blastocysts output was lower in GnRHa group (10.1% versus 27.4% in control group; P<0.05). In addition, re-expansion rates after vitrification, thawing and in vitro culture were lower in GnRHa treated ewes, although differences did not reach statistical significance (55.5% versus 74.1% in GnRHa treated and in control sheep, respectively).

Efficiency of superovulatory protocols is affected by the occurrence of reproductive abnormalities, such as the presence of anovulatory follicles. The objective of current study was to assess the incidence and possible causes of anovulatory follicles in superovulated sheep, in order to characterize the endocrine functionality of these follicles in terms of estradiol production and to evaluate their relationship with development of embryos from other follicles. The number and size of all follicles present in the ovaries of 12 sheep treated with a superovulatory FSH step-down treatment was assessed by ultrasonography. On Day 3 after subsequent estrus behaviour, the number of corpora lutea and anovulatory follicles were recorded and the fluid of anovulatory follicles >or=5mm in size was aspirated and assayed for estradiol. At once, embryos were recovered to evaluate their viability. In current study, anovulatory structures averaged 34.6% of the follicles developing to preovulatory sizes. The number of anovulatory follicles was determined by the existence of follicular dominance effects, since they increased with a higher difference in size between the largest and the second largest follicle at the beginning of the superovulatory treatment (P<0.05, r(2)=0.420). Most of the anovulatory follicles showed signs of functionality failures, indicated by a low mean estradiol concentration (9.9+/-1.1 ng/ml). However, a 22.4% of them were highly estrogenic (>200 ng/ml) and their permanence beyond the ovulation was related to a drop in the embryo viability rate (P<0.005), leading to decreased final superovulatory yields.

This study evaluated the effect of freezing-thawing procedures on the viability of sheep embryos cryopreserved at various developmental stages. The survival rates of frozen-thawed embryos were compared with non-frozen counterparts. Embryos were recovered from the oviduct and uterus, at different days of the early luteal phase, and were classified at six different developmental stages: 2- to 4-cell (n = 72), 5- to 8-cell (n = 73), 9- to 12-cell (n = 70), early morulae (n = 42), morulae (n = 41), and blastocyst (n = 70). For each early cleavage stage and blastocysts, approximately half of the embryos, were frozen immediately by slow freezing with an ethylene glycol-based solution. The remaining embryos were cultured to the hatched blastocyst stage. All morulae and compact morulae were frozen after recovery with the same protocol. Cryoprotectants were removed using 1M sucrose solution, and then warmed the embryos were cultured to the hatched stage in a standardized in vitro culture. Embryo developmental stage had a significant effect on the ability to hatch following freezing (P<0.0001). The cryotolerance of the embryos fitted a regression (r2 = 0.908), increasing linearly from 2- to 4-cell embryos (17.1%) to morula stage (46.3%) and in a quadratic regression from the morula to the blastocyst stage (83.7%). Frozen early cleavage stage embryos had a significantly lower viability than their fresh counterparts (23.1 vs 83.1%; P<0.0001), with a similar rate of viability between fresh or frozen blastocysts (92.5 vs 83.7%). In conclusion, early sheep embryos are very sensitive to freezing per se and the survival rates following conventional freezing improve as embryo developmental stage progresses.

The objective of this study was to characterize follicular development, onset of oestrus and preovulatory LH surge, and in vivo embryo yields of sheep superovulated after treatment with a single dose of 1.5mg of GnRH antagonist (GnRHa). At first FSH dose, ewes treated with GnRH antagonist (n=12) showed a higher number of gonadotrophin-responsive follicles, 2-3mm, than control ewes (n=9, 13.5+/-3.8 versus 5.3+/-0.3, P<0.05). Administration of FSH increased the number of >or=4mm follicles at sponge removal in both groups (19.3+/-3.8, P<0.0005 for treated ewes and 12.7+/-5.4, P<0.01 for controls). Thereafter, a 25% of the GnRHa-treated sheep did not show oestrous behaviour whilst none control sheep failed (P=0.06). The preovulatory LH surge was detected in an 88.9% of control ewes and 66.7% of GnRHa-treated sheep. A 77.8% of control females showed ovulation with a mean of 9.6+/-0.9 CL and 3.3+/-0.7 viable embryos, while ewes treated with GnRHa and showing an LH surge exhibited a bimodal distribution of response; 50% showed no ovulatory response and 50% superovulated with a mean of 12.2+/-1.1 CL and 7.3+/-1.1 viable embryos. In conclusion, a single dose of GnRHa enhances the number of gonadotrophin-dependent follicles able to grow to preovulatory sizes in response to an FSH supply. However, LH secretion may be altered in some females, which can affect the preovulatory LH surge and/or can weak the terminal maturation of ovulatory follicles.

This report offers the results of two experiments developed to test possible benefitial effects of the presence of corpus luteum (CL) on in vivo and in vitro sheep embryo production; using two different breeds treated with two different protocols by two different teams at two different centres. In the first trial, estrus was synchronized in 11 ewes with two doses of cloprostenol, 10 days apart. On day 1 after estimated ovulation, sheep were treated with progestagen sponges and superovulated with eight decreasing doses (26.4 units NIH-FSH-S1 x 3, 22.0 units x 2, and 17.6 units x 3) of ovine FSH injected twice daily. Ovulation rate and number of embryos obtained in vivo were compared to those from 12 control ewes without cloprostenol treatment. Presence of a CL improves the number of transferable embryos (7.4+/-0.6 versus 4.1+/-0.6 in control ewes, P < 0.05). The second trial investigated the effects of the presence of CL on embryos produced in vitro from six ewes bearing CL and six ewes without CL at start a superovulatory treatment consisting of 96 units of ovine FSH administered in four equal doses given every 12 h. There were not detected effects of the CL on the number and size of follicles or in the number, morphology and ability to resume meiosis of their oocytes. However, oocytes from ewes with CL showed higher rates of fertilization (73.5 versus 45.5%, P < 0.005), higher development to blastocyst (35.8 versus 19.3%, P < 0.01) and higher hatching rates after vitrification (80.0 versus 25.0%, P < 0.05).

Current study assessed differences in the response of sheep to estrus synchronization either by the administration of two doses of prostaglandin or by the insertion of an intravaginal progestagen sponge. The preovulatory follicular dynamics and estradiol secretion, the ovulatory response and progesterone secretion and the number and quality of embryos were studied in 27 ewes treated with two doses of 100 microg of cloprostenol, 10 days apart, and in 29 sheep treated with progestagen sponges for 14 days. Percentage of sheep responding to the synchronization treatments with signs of estrus behaviour was similar between both groups (81.5% versus 72.4%, respectively). The use of progestagen resulted in a higher diameter of the largest follicle (6.6+/-0.2 versus 5.9+/-0.2, P<0.05), and a lower number of small (6.7+/-0.3 versus 9.6+/-0.4, P<0.005) and total follicles (10.3+/-0.3 versus 12.9+/-0.4, P<0.005). However, mean plasma estradiol concentration during the follicular phase was higher in cloprostenol treated sheep (P<0.005). The mean ovulation rate was similar in both treatments (1.7+/-0.2 versus 1.7+/-0.3), but progesterone concentration during the early luteal phase was again higher in sheep treated with cloprostenol (P<0.05). The mean number of retrieved oocytes/embryos was very similar in both treatments (1.2+/-0.2 versus 1.4+/-0.2) and showed similar fertilization rates (70.6% versus 66.7%), but, although differences did not reach statistical significance, final viability rate was higher in cloprostenol than in progestagen treated ewes (58.9% versus 46.1%, P=0.07). Current results give new evidences supporting the negative effects of progestagens on the functionality of ovulatory follicles and support the development of new protocols for assisted reproduction including the use of prostaglandin analogues.

The current study assessed both the effects of in vitro culture and developmental stage of early stage in vivo produced ovine embryos on their ability to survive cryopreservation. Early stage embryos (n=226) were recovered from the oviduct, at different days of the early luteal phase, at three different developmental stages: 2- to 4-cell, 5- to 8-cell and 9- to 12-cell. For each stage, half of the embryos were cultured to the blastocyst stage and frozen thereafter (CF), while the remainder was frozen just after recovery (EF). A third experimental group (BF; n=43) included blastocysts obtained from the uterus and frozen immediately after recovery. Embryo viability post-thawing was determined by assessing their rate of development to the hatched blastocyst stage following in vitro culture. Culture negatively affected embryo viability, since survival rate was higher in blastocysts obtained from the uterus than in those from culture (83.7% versus 66.1%; P<0.05); also the cryosurvival of cultured embryos was lower when the timing of blastocyst formation was extended (P<0.01). However, survival following freezing-thawing of early developmental stages was significantly lower when compared to viability of their counterparts cultured to the blastocyst stage (23.1% versus 66.1%, P<0.0001). In conclusion, our results indicate that, despite the deleterious effects of culture per se, the culture of early in vivo produced ovine embryos to the blastocyst stage prior to be frozen improves their survival after thawing.

A total of 64 ewes was used to determine if the changes in superovulatory yields related to the ovarian status at the start of superovulatory treatment are due to differences in the population of gonadotrophin-responsive follicles, alterations in the processes of ovulation or transport of embryos from oviduct to uterus and/or developmental competence of the oocyte/embryo. Ovarian status at the start of a superovulatory FSH step-down treatment, administered coincidentally with a progestagen, was assessed by ultrasonography. On Day 4 after progestagen withdrawal, embryos were recovered from oviduct and their viability was determined by assessing development in vitro culture (IVC) until the hatched blastocyst stage. In all the ewes, the ovulation rate was related positively to the number of 2-3 mm follicles at first FSH injection (P<0.005). However, the total number of embryos and their viability were related to the more limited category of 3 mm follicles (P<0.05), whereas a higher degeneration rate was related to the number of 2mm follicles. The presence of a corpus luteum (CL) at the start of superovulatory treatment exerted a protective effect on embryonic viability, decreasing the degeneration of embryos. On the other hand, the presence of a dominant follicle at first FSH dose affected the mean size of the pool of follicles responding to the superovulation treatment, because ovulation arose from 3 to 5 mm follicles in absence of large follicles (P<0.05), but from 2 to 3 mm follicles when large follicles were present (P<0.005), indicating atresia in medium sized follicles in the presence of a large follicle.

The administration of growth hormone (GH) or GH plus GnRH antagonists (GnRHa) in sheep allows the enhancement of the pool of gonadotrophin-responsive follicles present in the ovaries and may be useful to increase yields obtained in embryo programmes. The objective of the current study was to evaluate the ability of follicles recruited in response to treatment with GH and GnRHa to grow in response to exogenous follicle stimulating hormone (FSH) and the competence of their oocytes to resume meiosis. Seven females were treated with two doses of GnRHa (days 0 and 3) and three doses of 15 mg of GH (days 3, 4 and 5). Thereafter, this group and a second group (n = 7) were treated with three doses of 1.5 ml of FSH 12 h apart. A third group (control; n = 4) did not receive GH/GnRHa or FSH. The mean number of follicles aspirated on day 7 was higher in ewes treated with GH and GnRHa prior to the stimulation with exogenous FSH than in ewes treated with FSH without pretreatment and in untreated control sheep (20.4 +/- 2.6 vs 17.7 +/- 3.9 and 11.5 +/- 0.8, p < 0.05 and p < 0.01, respectively). The number of recovered cumulus-oocyte complexes after follicular aspiration was higher in the GH/GnRHa + FSH group (8.7 +/- 0.9 vs 6.8 +/- 1.3 in FSH group, n.s., and 4.5 +/- 0.8 in control, p < 0.05), but there were no differences found in the resumption of meiosis (63.1 +/- 9.5% for GH/GnRHa + FSH vs 79.5 +/- 6.3% for FSH and 60.0 +/- 8.8% for control). These results indicate that GH and GnRHa would be useful to increase the number of gonadotrophin-responsive follicles in the ovary, but adjustment of later FSH treatment allowing further development of follicles may be necessary prior to its use in superovulatory protocols.

The objective was to study the endocrine activity in sheep with large ovarian follicles and the effects of dominant follicles on other follicles, looking for possible intraovarian differences. Induction of dominant follicles was achieved using controlled exogenous LH pulses every 90 min over 14 days in eight Scottish Blackface ewes. During this period, follicular development was assessed by daily transrectal ultrasonography and jugular venous blood samples were collected every 12 h for FSH, LH inhibin and oestradiol assay. The exogenous LH pulses caused the appearance of large follicles in all the ewes, which reached a maximum mean diameter of 7.2 +/- 0.5 mm on Day 5.5 +/- 2.6 after first detection. In the presence of a dominant follicle, no other follicle grew to a diameter larger than 4 mm and there was a decrease in the number of new growing follicles (P < 0.05) and in the number of smaller follicles (P < 0.01). This effect of dominance was mediated by changes in FSH concentration, since FSH level decreased (P < 0.05) as dominant follicles grew and the decrease in FSH levels was related to a decline in the number of remaining follicles (P < 0.05). However, the greatest decrease in the number of small follicles growing to larger sizes was observed in the ovary ipsilateral to the dominant follicle (P < 0.05). These data confirm that the presence of a large follicle depresses the recruitment and growth of other follicles by systemic factors and provide some evidence of local inhibitors blocking the final development of other putative large follicles.

It has been suggested that ewes carrying the Booroola gene (Fec(B)) consistently ovulate more follicles because they recruit more primordial follicles and/or have a lower rate of atresia. If the former is correct, the pool of follicles would be depleted sooner in Fec(B) animals. We have studied follicular dynamics and endocrine function during follicular and early luteal phases of the estrous cycle of older ewes with or without the fecundity gene and compared this data with data obtained 6 yr previously in the same animals. Older sheep carrying the Booroola gene maintained a significantly higher ovulation rate than noncarrier ewes [4.2 +/- 0.8 vs. 2.2 +/- 0.6 corpora lutea (CL), respectively; P < 0.05], and in keeping with data from young animals, both ovulatory follicles and CL (4.7 +/- 0.3 vs. 6.9 +/- 0.7 mm and 12.8 +/- 0.5 vs. 16.7 +/- 0.8 mm, respectively) were smaller than those of noncarrier ewes (P < 0.05). The interval from luteolysis to the onset of the LH surge increased with age in all the animals (from 52.0 +/- 8.0 to 67.0 +/- 7.5 h in gene carrier sheep and from 56.0 +/- 2.0 to 79.5 +/- 9.6 h in noncarrier sheep, P < 0.05). The concentration of estradiol and inhibin A in the early luteal phase was lower in older noncarrier ewes (P = 0.08 and P < 0.05, respectively), and the level of inhibin A was inversely related to the level of FSH in aged sheep of both genotypes (P < 0.0001). In contrast, the number of developing follicles in older ewes of both genotypes was similar to the number found in younger ewes, suggesting that increased ovulation rate in sheep carrying the Fec(B) mutation is related to a reduced rate of atresia.

The main objective of this study was to compare the effect of the presence of large follicles at the start of FSH treatment on the superovulatory response in ewes in the breeding and nonbreeding seasons. A second objective was to verify the effect on the superovulatory response of the presence of a corpus luteum at the start of the FSH treatment during the breeding season. Fifteen ewes in breeding season (October) and 14 in nonbreeding season (May-June) were treated with 40 mg FGA sponges (Chronogest) for 14 days, together with a single dose of 125 microg cloprostenol on Day 12, considering Day 0 as day of progestagen insertion. Superovulatory treatments consisted of eight decreasing doses (1.5 ml x 3, 1.25 ml x 2 and 1 ml x 3) of Ovagen twice daily from 60 h before to 24h after sponge removal. Ovarian structures were assessed by transrectal ultrasonography using a 7.5 MHz linear array probe. Luteal activity at progestagen insertion (Day 0) and presence of corpus luteum and of large follicles at first FSH dose (Day 12) were determined. There were no significant differences between the breeding season and nonbreeding season for ovulation rate (11.6+/-1.4 versus 11.6+/-1.3), number of recovered embryos (8.0+/-1.1 versus 9.6+/-1.3) or number of viable embryos (7.2+/-1.1 versus 5.8+/-1.2). During the breeding season, there were fewer recovered embryos in ewes with a large follicle (> or =6mm) at first FSH dose (6.9+/-1.1 versus 12.3+/-1.8, P<0.05) and fewer viable embryos (5.0+/-1.2 versus 10.5+/-0.5, P<0.05) than in ewes without such a follicle. During the nonbreeding season, however, there were no significant differences between ewes with or without a large follicle for either recovered (9.0+/-2.5 versus 11.3+/-1.2) or viable embryos (6.3+/-2.3 versus 8.1+/-1.2). Analysis of seasonal differences in ewes with a large follicle showed a lower number of recovered embryos in the breeding season (P<0.05) due to a lower recovery rate (65.7% versus 92.3%, P<0.05), since mean number of corpora lutea in response to the FSH treatment was similar (10.9+/-1.3 versus 10.0+/-2.5). These results indicate that, in sheep, the inhibitory effects of large follicles during the nonbreeding season are not as obvious as during the breeding season.

Objectives of this study were to characterize patterns of follicular development in sheep superovulated with purified follicle stimulating hormone (FSH) (OVAGEN, ICP, Auckland, New Zealand) and to determine its influence on preovulatory events (onset of the oestrus behaviour and timing of the preovulatory luteinizing hormone surge) and ovarian response (ovulation rate and embryo yield). Number and size of all >/= 23 mm follicles from the first FSH injection to withdrawal of progestagen sponges was determined by transrectal ultrasonography just prior to every FSH injection in nine Manchega ewes superovulated with eight decreasing doses (ml) (1.5 x 3, 1.25 x 2 and 1 x 3) of OVAGEN injected twice daily from 60 h before to 24 h after the withdrawal of 40 mg fluorogestone acetate sponges. Oestrous detection and jugular blood sampling for LH radioimmunoassay were performed every 3 h from 14 to 53 h after sponge removal and ovulation rate and number of embryos were determined 4 days after progestagen withdrawal. Administration of OVAGEN induced a significant rise (p < 0.0005) in the number of follicles >/= 4 mm in size because of an increased growth in size of follicles from the first FSH injection to sponge removal, an increase in the number of newly detected follicles from 12 to 36 h of the first FSH dose (p < 0.005) and a decrease in regression rate from 24 h (p < 0.001). The number of follicles 2-3 mm in size at first FSH dose (10.4 +/- 1.5) was positively correlated with the number of >/= 4 mm follicles at 0 h (19.0 +/- 2.7, p < 0.01). A higher number of >/= 4 mm follicles at 0 h was related with an earlier appearance of oestrus (31.5 +/- 1.5 h, p = 0.08) and LH surge (45.0 +/- 2.3 h, p < 0.005), and a higher ovulation rate (18.2 +/- 3.8, p < 0.005). On the other hand, the rate of embryo recovery was decreased in ewes with earlier preovulatory LH peaks (p < 0.005), with a shorter interval between oestrus and LH peak (p < 0.05).

The present study was developed to assess possible effects on ovulatory response and embryo yields arising from the presence of a corpus luteum (CL) at the time of initiation of the progestagen treatment used in superovulatory protocols in sheep. In breeding season, estrus was synchronized in 25 Manchega ewes using 40 mg FGA sponges for 14 days, together with a single dose of 125 microg of cloprostenol on Day 12, with Day 0 as day of progestagen insertion. Superovulatory treatment consisted of eight decreasing doses (1.5 x 3 ml, 1.25 x 2 ml, and 1 x 3 ml) of Ovagen twice daily from 60 h before to 24 h after sponge removal. The presence or absence of corpora lutea was assessed by transrectal ultrasonography at progestagen insertion and at first FSH dose. Number and size of all follicles > or = 2 mm were also evaluated at first FSH dose. The number of corpora lutea and the number and viability of recovered embryos in response to the treatment were evaluated 7 days after sponge removal. No significant effect on ovarian response of the presence of a CL at sponge insertion in 21 of the 25 ewes (84%) was detected. However, ewes with a CL at first FSH dose (16 ewes, 64%) yielded a higher number of transferable embryos (7.2 +/- 1.4 versus 2.7 +/- 0.7, P < 0.05), since the embryo degeneration rate was increased in sheep without a CL (42.5% versus 12.7%, P < 0.01). Analysis of possible effects derived from the presence of a large presumptively dominant follicle (> or = 6 mm) at first FSH dose showed that both recovery and viability rates were lowest (P < 0.05) in ewes bearing a large follicle in the absence of a CL (40.5 and 50.6%, respectively), and highest in ewes that did not show a large follicle but in which a CL was present (73.9 and 85.2%). The final number of transferable embryos was very different between groups (10.2 versus 1.8, P < 0.01). These results indicate that the number and quality of embryos obtained from superovulated ewes is affected by the presence of a CL prior to the first FSH dose (i.e. by the stage of the estrous cycle at progestagen insertion) and also by an interaction with suppressive effects from large dominant follicles. This finding suggests the existence of some effects on follicular population prior to the FSH treatment that may compromise follicle and oocyte developmental competence. It seems reasonable to hypothesize that superovulatory yields would be increased by beginning the treatment during the early-luteal phase of the estrous cycle, allowing for the presence of a CL along with the progestagen treatment.

Variability in superovulatory response to FSH stimulation is common to most mammals and imposes practical problems for assisted reproduction. In sheep, we have studied if this response is related to the ovarian follicular population and activity before the stimulation. During the breeding season, 30 ewes were treated with 40 mg FGA sponges for 14 days and 125 microg cloprostenol injection on Day 12, considering Day 0 as the day of progestagen insertion. Superovulatory response was induced with two different FSH regimes using the same total dose (8.8 mg), administered twice daily from 60 h before to 24 h after progestagen withdrawal. At the first FSH injection, all follicles > or = 2 mm were observed by transrectal ultrasonography and plasma FSH and inhibin A levels were determined. The number of corpora lutea and the number of and viability of recovered embryos in response to the treatment were determined on Day 7 after sponge withdrawal. No significant differences were found between treatments. The total mean number of corpora lutea (11.5 +/- 1.2) and recovered embryos (7.9 +/- 1.1) were positively correlated (P < 0.05 and <0.01, respectively) with the number of small antral follicles (2-3 mm: 9.2 +/- 0.7) and inhibin A concentration (240 +/- 18 pg/ml; P < 0.05 for corpora lutea and P < 0.005 for recovered embryos) observed at the onset of the superovulatory treatment, which was also positively correlated with the number of viable embryos (5.8 +/- 0.9, P < 0.005). In 18 ewes with follicles > or = 6 mm prior to FSH treatment, the ovulation rate was unaffected but the number of embryos (6.1 +/- 0.9 versus 11.6 +/- 2; P < 0.05) and their viability (4.5 +/- 0.8 versus 8.5 +/- 2; P < 0.05) was reduced. The lower number of embryos produced when a large follicle is present suggest that a proportion of the smaller follicles are in early stages of atresia and the developmental competence of their oocyte is compromised.

The timing of the onset of puberty, duration of seasonal ovulatory activity, and length of postpartum anestrus were studied by means of blood plasma progesterone concentrations in a flock of European female mouflons (Ovis gmelini musimon) maintained in captivity under natural photoperiod (40 degrees 25'N). Concentrations of progesterone in the peripheral blood were determined by radioimmunoassay in samples collected from the jugular vein twice a week. First ovulations in the breeding season were highly synchronized and occurred in mid-October. In contrast, the cessation of ovulatory cycles showed significant variation among females and extended from February to May, depending on age, with 2-yr-old animals exhibiting the longest anovulatory period (P < 0.01). When lambing occurred within the breeding season (February-April), 12 out of 26 animals had their first ovulation 25 +/- 1.8 days after parturition. The 14 late-lambing females had the first postpartum ovulation delayed until the next breeding season. March/April-born mouflon lambs that reached a minimum threshold body weight (23.8 +/- 0.6 kg) in their first breeding season reached puberty at 8 mo of age. In those with slower growth rates, however, the prepubertal period was extended throughout the first breeding and nonbreeding seasons, reaching puberty during the breeding season of the following year at 19 mo of age and 27 +/- 0.3 kg body weight. Further, attainment of puberty in ewe lambs born in June/July was also delayed until the breeding season of the following year, when animals had reached a threshold body weight at 17 mo of age.

Daily transrectal ultrasonographies were conducted to study development of all follicles with antral diameters > or = 2mm during the follicular phase of oestrous cycle in Mouflon, a strictly monovular wild-sheep. A total of 14 follicular phases was studied after oestrus synchronization with two cloprostenol doses, 9 days apart, in five cyclic Mouflon ewes. In 13 cycles (92.8%), the ovulatory follicle arose from those antral follicles present in both ovaries when luteolysis was induced, being the largest one with a mean size of 4.4+/- 0.3mm at that moment in 10 cycles (76.9%). The remaining cycles had a larger follicle, but it was decreasing in size. Appearance of new follicles > or =2mm in size remained unaffected during the follicular phase (3.7+/- 0.2), but there was found a linear decrease in the number of those growing to > or =3mm (2.5+/- 0.4 to 1.1+/- 0.2, P < 0.05) and > or = 4mm (0.6+/- 0.2 to 0.1+/- 0.1, P < 0.005), detection of new follicles growing to > or = 5mm was negligible. Then, number of medium (4-5mm) growing follicles present in both ovaries decreased from 1.5+/- 0.3 at 0 h to 0.3+/- 0.1 at 72 h (P<0.005). In conclusion, the single ovulatory follicle is the largest growing follicle present in both ovaries at the moment of luteolysis. This follicle is selected to grow and ovulate while development of other follicles is inhibited.