Pessary Withdrawal Research Articles

Superovulation of Boer goats with different synchronization regimens at different times of the year in the northern temperate zone

The present study addresses a comparison of various estrous cycle control protocols and the effect of season in the northern temperate zone on the superovulatory response in Boer goats. Four groups of 12 does each were treated in February, May, August and November, respectively. Does were provided with progestogen containing intravaginal pessaries to synchronize estrus. Half the does of each group received sponges impregnated with 20 mg fluorogestone acetate (“Cronolone”), the other half “Eazy-Breed” CIDRs containing 0.3 mg progesterone. Pessaries remained in place for 7 days. Within each subgroup half the does were treated with PGF2α (“Dinoprost”) at pessary insertion, the other half at pessary withdrawal. Beginning 48 h before pessaries were removed does received i.m. injections of 4, 4, 2, 2, 2 and 2 Armour Units (AU) FSH, supplemented with 40% LH, at 12 h intervals. Estrus detection was conducted at 8 h intervals and ovarian activity was monitored by daily transrectal ultrasonography. Does were mated and 7 days later non-surgical embryo collection was conducted. It transpired that the type of intravaginal pessary had no effect on the time passing between pessary withdrawal and onset of estrus (sponge: 40.7 h vs. CIDR: 35.2 h), number of ovulations/doe assessed by echographic counting of collapsed large follicles (7.2 vs. 7.6) or corpora lutea (7.1 vs. 9.8), embryo recovery rate (39% vs. 41%) and proportion of transferable embryos (66% vs. 65%). The number of collapsed large follicles was significantly higher than that of corpora lutea on the day before embryo collection (7.4 vs. 8.5; P < 0.05), the correlation between the two being r = 0.55 (P < 0.05). The effect of PGF2α administration at the onset or at the end of progestogen treatment and the effect of season were not significant, except for a shorter interval between pessary removal and onset of estrus in August in comparison to the other months (29.3 vs. 39.4, 40.6 and 42.1 h; P < 0.05). In conclusion, Boer goats, although being seasonal breeders in the northern temperate zone, will respond to superovulatory treatment and produce embryos of high quality year round regardless of type of pessary and time of PGF2α administration.

Effects of diet type on establishment of pregnancy and embryo development in beef heifers

The objectives were to determine the effects of elevated blood urea concentrations on: (i) the response to superovulation, fertilisation rate, and early embryonic development in beef heifers, and (ii) embryo survival from days 7 to 35 of gestation. In Experiment 1, heifers (18–24 months) were allocated at random (n=20 per treatment) to one of the following diets: (i) ad libitum grass silage plus 5kg commercial beef concentrates per day (controls); (ii) ad libitum grass silage plus 5kg concentrates and 250g feed grade urea per day (HE/HU); or (iii) ad libitum wheaten straw plus 250g feed grade urea and 50g vitamin/mineral mix per day (LE/HU). Serum urea concentrations were monitored throughout the experiment. Oestrus in heifers was synchronised using an intravaginal releasing device (CIDR®, InterAg, New Zealand). Oestrus was detected and in vitro produced blastocysts (day 7, morphological grades 1 and 2) were transferred to the heifers 7 days later (19 days after start of treatment diets). The heifers were maintained on the dietary treatments for a further 28 days, when pregnancy status was determined by transrectal ultrasonography. Detected pregnancies were terminated using 15mg luprostiol and recycled for Experiment 2. In Experiment 2, following a 14-day dietary rest period, the heifers were re-allocated at random to the three dietary treatments above. Heifers were treated with a CIDR for 8 days and 15mg luprostiol was given 12h before pessary withdrawal. They received 144mg pFSH (Folltropin®-V, Vetrepharm, Canada) given as 8 injections over 4 days commencing on day 6 of CIDR/dietary treatment. Heifers were artificially inseminated 48h after progesterone pessary withdrawal using commercial semen of proven fertility by a competent inseminator. The heifers were maintained on their diets until slaughter, 3 days post insemination when corpora lutea numbers were determined and embryos were recovered and cell numbers determined visually. Serum urea concentrations were greater in heifers on LE/HU than in those on HE/HU diets, which in turn were greater than controls (7.1±0.5, 4.9±0.3 and 3.2±0.1mmol/L, respectively; P<0.05). There was no effect of diet type on pregnancy rate at day 35 (42%, 47% and 46%) and on the number of corpora lutea following superovulation (5.2±0.8, 5.8±1.5 and 6.8±1.1) for heifers on control, HE/HU and LE/HU diets, respectively. The total number of embryos recovered per heifer was not different between the three groups (2.7±0.6, 3.4±1.1 and 4.8±0.8 for heifers on control, HE/HU and LE/HU diets, respectively; P>0.05), but the number of embryos with 8 or more cells at recovery was greater in heifers on LE/HU than on control diets (3.4±0.8 compared with 1.0±0.3; P<0.05). However the percentage of embryos recovered with 8 or more cells was not different between groups (70.0±13.3, 86.9±7.2 and 76.5±7.9%, for heifers on control, HE/HU and LE/HU diets respectively). Fertilisation rate, expressed as the proportion of embryos with more than one cell at recovery relative to the total number of embryos recovered, was less in the heifers on the control diet than in the other two dietary treatments (61.3±11.8, 92.0±3.5 and 86.8±5.4% for heifers on control, HE/HU and LE/HU diets, respectively; P<0.05). Deleterious effects of urea on reproduction were not found, suggesting that adverse effects of urea are likely to take place at the early oocyte development stage prior to ovulation or fertilisation following an increase in protein intake.

Effect of insulin on the relationship of estrous behaviors to estradiol and LH surges in intact ewes

The objective of this study was to determine whether acute stress alters the frequency of spontaneous estrous behaviors and temporal relationships with preovulatory increases in peripheral plasma estradiol and LH. Follicular phases of intact ewes were synchronized with prostaglandin administered at progesterone pessary withdrawal (PW). Twelve ewes served as controls and 12 were acutely stressed (insulin 4 IU/kg given at 30 and 32 h after PW). Ewes being near to ram(s) 21.3 ± 1.9 h after PW was the first precopulatory behavioral sign and rams nosed the perineal region of ewes after a further 9.0 ± 2.0 h ( P < 0.01), with ewes being nudged and mounted by rams 6.8 ± 2.3 h later still ( P < 0.01). Insulin did not affect the frequency or timing (relative to PW) of each behavioral sign of estrus. However, within each animal, estradiol values were more than 2 pg/ml lower ( P < 0.05) for 6 h following insulin, and the onset of the LH surge was delayed in insulin-treated ewes compared to controls (49.5 ± 3.3 versus 38.2 ± 2.6 h; P = 0.01). Consequently, the interval between the onset of being mounted and the LH surge was longer in insulin-treated ewes compared to controls (10.4 ± 3.0 versus 2.3 ± 0.7 h; P < 0.01). Maximum LH values were also 15 ng/ml lower after insulin ( P < 0.01). Thus, acute stress did not alter the timing or frequencies of estrous behaviors but it did reduce estradiol concentrations and delayed the onset and magnitude of the LH surge.

407 SUPERSTIMULATORY RESPONSE WITH DECREASING DOSES OF FOLLICLE-STIMULATING HORMONE v. A SINGLE DOSE OF FOLLICLE-STIMULATING HORMONE - EQUINE CHORIONIC GONADOTROPIN DURING BREEDING AND NONBREEDING SEASON IN SHEEP

The aim of the study was to assess possible variations in superovulatory yields due to different FSH treatments at 2 times of the year. Superovulation and embryo recovery were performed during the breeding (n = 63) andnonbreeding (n = 46) seasons in Merino ewes located at 41°S latitude. Animals were kept under the same conditions, housed outdoors in a sheltered and covered pen, and were fed a liveweight maintenance ration. All animals received 60-mg medroxyprogesterone acetate intravaginal sponges (Progespon®, Syntex, Buenos Aires, Argentina) for 14 days. Ewes were then randomly assigned to 2 different superovulatory treatments: classic (n = 74) and one shot (n = 35) in both seasons. Classic superovulatory treatment consisted of 7 decreasing doses (2 × 48 mg, 2 × 24 mg, 2 × 20 mg, and1 × 16 mg NIH-FSH-P1)ofFSH (Folltropin®-V, Bioniche, Belleville, Ontario, Canada), administered twice daily from 48 h before to 24 h after pessary removal. A dose of eCG (300 IU; Novormon®, Syntex) was administered at progestagen removal. One shot superovulatory treatment consisted of a single dose of FSH (70 mg NIH-FSH-P1) plus 300 UI of eCG injected at pessary withdrawal. Embryo donors were inseminated by laparoscopy with frozen-thawed semen (100 × 106 spz) 12 h after the onset of estrus. Surgical embryo recovery was done on Day 7 after sponge withdrawal and embryos were graded for quality according to morphology (Grade 1 = excellent or good; Grade 2 = fair; Grade 3 = poor; and Grade 4 = dead or degenerated; IETS 1998). A 2 × 2 factorial ANOVA was used to test the main effects (season and superovulatory treatment) and interactions. There were no significant differences in the proportion of responding ewes (&gt;3 corpora lutea), ovulation rate, and recovered Grades 1 to 2 embryos between the breeding and nonbreeding season (Table 1; P &gt; 0.05). However, number of recovered ova/embryos and ova/embryo recovery rate were higher during the breeding season compared with the nonbreeding season, whereas the percentage of nonfertilized ova was lower in the breeding season than in the nonbreeding season (P &lt; 0.05). Analysis of data comparing superovulatory treatments showed that the proportion of responding ewes, ovulation rate, recovered embryos, and recovered Grades 1 to 2 embryos were lower for the one shot treatment than for the classic treatment (P &lt; 0.05). Embryo recovery rate and nonfertilization rate did not differ between treatments (P &gt; 0.05). It was concluded that there was an increase in the number of total recovered ova/embryos during the breeding season compared with the nonbreeding season, although the number of recovered good-quality embryos was not affected. The use of multiple FSH injections produced a higher number of total recovered and viable embryos in Merino sheep than the one shot superovulatory treatment. Table 1.Embryo yields in ewes submitted to superovulation

A new strategy for superior reproductive performance of ewes bred out-of-season utilizing progestagen supplement prior to withdrawal of intravaginal pessaries

Two experiments were conducted to examine the effect of progestagen supplement 24h prior to intravaginal pessary withdrawal on reproductive performance of seasonal anestrous ewes. Ewes in each experiment were allocated to treatment and control and all were induced to estrus using either intravaginal MAP (Exp. 1; n=24) or CIDR-G (Exp. 2; n=28) pessaries for 12 days. Half of the ewes in each experiment were supplemented 24h before withdrawal of pessaries with either 10mg oral MAP tablets (Exp. 1) or 25mg i.m. progesterone (P(4)) administration (Exp. 2; P(4)-supplement-treated group). Fertile rams were allowed with the ewes at sponge removal (Day 0, 0h) and estrus was monitored at 6-h intervals for 3 days. Blood samples were collected for measurements of P(4) (Exp. 1 and Exp. 2) and LH (Exp. 2). In both experiments, the percent of ewes in estrus was greater (P<0.05) and intervals to estrus were longer (P<0.05) in progestagen-supplement-treated than control ewes. In Exp. 2, the occurrence and magnitude of LH surges were greater (P<0.01) and intervals to onset of LH surge were longer (P<0.01) in P(4)-supplement-treated than control ewes. In Exp. 2, P(4) supplement elevated P(4) levels from 1.8+/-0.1ng/mL on Day -1 to 4.2+/-0.3 on Day 0 (P<0.001). Following pessaries removal, P(4) concentrations fell to basal values on Day 1 in both groups and remained low until Day 5. Then, P(4) concentrations increased and remained elevated through Day 19 in all (100%) progestagen-supplement-treated in Exp. 1 (12/12) and Exp. 2 (14/14) and in only 5/12 (41.7%) and 6/14 (42.9%) control ewes, respectively. These ewes were confirmed pregnant by ultrasonography and lambed on Day 149.2+/-0.2 following Day 0. In conclusion, progestagen supplement 24h prior to removal of pessary can be used successfully to improve reproductive performance of ewes bred out-of-season.

Simplified superovulatory treatments in Corriedale ewes

Two experiments were designed to evaluate the possibility of simplifying superovulatory treatments in Corriedale ewes with use of ovine FSH (oFSH). Ewes received intravaginal progestogen sponges for 14 days. In Experiment 1, several simplified schedules were tested. Ewes were treated with 176 NIH-FSH-S1 units’ oFSH given as a single injection in saline, along with 500 IU eCG 48 h before sponge removal (Group A1), in four equal doses (B1), or given as a single injection in a polyvinylpyrrolidone vehicle (C1) 24 h before sponge removal. In Experiment 2, the simplified protocol that exhibited the most desirable results in Experiment 1 (A2) was compared with the same protocol, but using less oFSH (132 units) (B2) and with the most conventional protocol (176 units of oFSH in eight decreasing doses; C2). Estrus was detected and ewes were naturally mated. The ovarian response and embryo production were assessed on Day 6 after estrus. LH was measured at 6 h intervals from pessary withdrawal. The onset of estrus and the pre-ovulatory LH surge were advanced ( P < 0.05) in ewes treated with FSH and eCG. In Experiment 1, protocol A1 produced a greater percentage of superovulated ewes compared to C1 (100.0 compared with 58.3%; P < 0.05), increased ovulation rate (13.8 corpora lutea compared with 6.2 and 4.7 for B1 and C1, respectively; P < 0.05), and tended to increase the number of transferable embryos compared to B1 ( P = 0.08). In Experiment 2, percentages of superovulated ewes and ovulation rates were similar among groups; however, Group A2 tended to have more large follicles ( P = 0.07) than C2. The number of transferable embryos was similar among the three treatments. In conclusion, the reduced-dose oFSH given once along with eCG is the most appropriate superovulatory treatment because it combines simplicity and a lesser dose of gonadotropin, which also implies a reduction in cost, without reducing embryo production.

A note on mating preferences in Rasa Aragonesa rams

Two experiments were conducted to determine whether Rasa Aragonesa rams, a Mediterranean breed, mount ewes randomly or preferentially when presented with a group of 12 oestrous-synchronized ewes during the period of natural seasonal anoestrus. In the first experiment, 12 Rasa Aragonesa ewes were induced into oestrus using intravaginal pessaries. Forty-eight hours after pessary withdrawal, the group of ewes was presented to an aproned ram (to prevent ejaculation) and the time spent by the ram courting and mounting each ewe was recorded. To quantify differences in sexual attractiveness among ewes, a modified Tillbrook test was performed. The procedures were repeated using eight rams. Both the average amount of time spent by rams courting individual ewes (15.4 s, S.E. = 2.9, range of means = 11.7–22.5 s) and the average amount of time that individual ewes were courted by an individual ram (15.4 s, S.E. = 3.5, range of means = 9.7–21.7 s) varied significantly ( P < 0.05). In the second experiment, each of 5 rams was presented to a group of 12 oestrous-synchronized ewes. Rams were permitted to court and service ewes over 2 h periods and individual ewes were not removed after being serviced. The number of services per ewe was recorded. Rams differed significantly in the number of ewes serviced. Collectively, these results suggest that mating management techniques should appreciate that minimizing the ram-to-ewe ratio could foster lower fecundity in Rasa Aragonesa sheep.

Embryo survival and recipient pregnancy rates after transfer of fresh or vitrified, in vivo or in vitro produced ovine blastocysts

The aim of this study was to assess the effect of production system and of cryopreservation of ovine embryos on their viability when transferred to recipients. The experimental design was an unbalanced 2×2 factorial design of two embryo production systems (in vivo versus in vitro) and two embryo preservation conditions prior to transfer (transferred fresh versus transferred after vitrification/warming). For the production of blastocysts in vivo, crossbred donor ewes ( n=30) were synchronised using a 13-day intravaginal progestagen pessary. Ewes received 1500 IU equine chorionic gonadotropin (eCG) 2 days before pessary withdrawal, and were mated 2 days after pessary withdrawal and embryos were recovered surgically (6 days after mating). Blastocysts were produced in vitro (IVP) using standard techniques. Recipients ( n=95) were synchronised using a progestagen pessary and received 500 IU eCG at pessary removal and were randomly assigned to receive (two per recipient) in vivo fresh ( n=10), in vivo vitrified ( n=10), in vitro fresh ( n=35) or in vitro vitrified ( n=40) blastocysts. Recipients were slaughtered at day 42 of gestation and foetuses recovered. Pregnancy and embryo survival rates were recorded and analysed using CATMOD procedures. Foetal weights and crown-rump lengths were recorded and analysed using generalised linear model (GLM) procedures. There were no statistically significant interactions between the effects of embryo production system and preservation status at transfer on pregnancy rate and embryo survival. The pregnancy rate following transfer of fresh IVP blastocysts was lower ( P<0.07) than that of in vivo embryos (54.3% versus 90.0%, respectively). Vitrification resulted in a decrease in pregnancy rate, the effect being more pronounced in the case of IVP embryos (54.3–5.0%, P<0.001) compared with in vivo embryos (90.0–50.0%), although the absolute change was similar (49.3% versus 40.0%). Transfer of fresh IVP blastocysts resulted in a higher proportion of single (78.9% versus 33.3%) and lower proportion of twin (21.1% versus 66.7%) pregnancies than those produced in vivo. This was reflected in a significant difference in embryo survival rate (fresh: 32.8% versus 75.0%, P<0.01; vitrified: 2.5% versus 35.0%, P<0.001, for IVP and in vivo blastocysts, respectively). Similarly, all pregnancies resulting from the transfer of vitrified/warmed IVP blastocysts were single pregnancies, while 40% of those from vitrified/warmed in vivo blastocysts were twin pregnancies; this was reflected in an embryo survival rate of 35.0% versus 75.0%, respectively. There was a significant effect ( P=0.0184) of litter size on foetal weight but not on foetal length ( P=0.3304). Foetuses derived from the fresh transfer of IVP blastocysts were heavier (6.4±0.2 g versus 5.8±0.2 g, respectively, P<0.05) and longer (5.2±0.1 cm versus 4.8±0.1 cm, respectively, P<0.01) than those derived from fresh in vivo blastocysts. There was no difference in these parameters as a consequence of vitrification of IVP embryos. However, in vivo blastocysts subjected to vitrification resulted in heavier (6.6±0.3 g versus 5.8±0.2 g, respectively, P=0.055) and longer (5.2±0.1 cm versus 4.8±0.1 cm, respectively, P<0.05) foetuses than their counterparts transferred fresh.

Interleukin 8 in the cervix of non-pregnant ewes

Studies of cervical artificial insemination of ewes at hormone-synchronized oestrus indicate that the cervix remains relatively impenetrable to semen, in contrast to naturally breeding animals. During parturition the inflammatory response plays an important part in cervical dilation and possibly, to a lesser extent, in the non-pregnant cervix at oestrus to facilitate the transcervical transport of semen. The expression of interleukin 8 (IL-8), a pro-inflammatory cytokine in the ovine cervix, has been mapped and quantified, using semi-quantitative in situ hybridization, to ascertain the role played by inflammation in the ovine cervix during natural and artificially induced oestrous cycles. IL-8 gene expression was observed in both the luminal epithelium and fibroblastic cells of the cervix. The presence of IL-8 was confirmed using immunohistochemistry. IL-8 gene expression in the luminal epithelium varied throughout the oestrous cycle and was highest at oestrus and at day 5 of the oestrous cycle. In ewes artificially induced to ovulate, either by the withdrawal of progesterone pessaries after treatment for 12 days, or by two i.m. injections of prostaglandin 9 days apart, IL-8 gene expression at oestrus was significantly lower than it was at natural oestrus. Insemination increased IL-8 gene expression in progesterone-synchronized ewes. These data support the hypothesis that IL-8-induced inflammation is important in normal cervical function and that this process is inhibited during artificial synchronization of the oestrous cycle and is increased by exposure to semen.

Interleukin 8 in the cervix of non-pregnant ewes

Studies of cervical artificial insemination of ewes at hormone-synchronized oestrus indicate that the cervix remains relatively impenetrable to semen, in contrast to naturally breeding animals. During parturition the inflammatory response plays an important part in cervical dilation and possibly, to a lesser extent, in the non-pregnant cervix at oestrus to facilitate the transcervical transport of semen. The expression of interleukin 8 (IL-8), a pro-inflammatory cytokine in the ovine cervix, has been mapped and quantified, using semi-quantitative in situ hybridization, to ascertain the role played by inflammation in the ovine cervix during natural and artificially induced oestrous cycles. IL-8 gene expression was observed in both the luminal epithelium and fibroblastic cells of the cervix. The presence of IL-8 was confirmed using immunohistochemistry. IL-8 gene expression in the luminal epithelium varied throughout the oestrous cycle and was highest at oestrus and at day 5 of the oestrous cycle. In ewes artificially induced to ovulate, either by the withdrawal of progesterone pessaries after treatment for 12 days, or by two i.m. injections of prostaglandin 9 days apart, IL-8 gene expression at oestrus was significantly lower than it was at natural oestrus. Insemination increased IL-8 gene expression in progesterone-synchronized ewes. These data support the hypothesis that IL-8-induced inflammation is important in normal cervical function and that this process is inhibited during artificial synchronization of the oestrous cycle and is increased by exposure to semen.

The effect of progestagen treatment on sheep reproductive performance at different phases of the oestrous cycle

Two experiments were carried out in order to determine the effect of progestagen treat- ment to synchronise oestrus on sheep reproductive performance at different phases of the cycle, com- paring artificial insemination and natural mating. In experiment 1, and after analysis of plasma progesterone concentrations 7 days before and at pessary insertions, animals were divided into four groups: Group F (n = 25), follicular phase at pessary insertion; Group L1 (n = 22), the onset of the luteal phase; Group L2 (n = 18), mid-late luteal phase and Group A (n = 11), anovulatory. The ewes were inseminated 54 h after pessary withdrawal. Mean percentage of fertility was low (30%), and when data from Groups L1 and L2 were pooled, and compared with F+A groups, percentage of fertil- ity was significantly higher (P < 0.05) in the latter groups (20% vs. 39%, respectively). Similarly, the number of lambs born per inseminated ewe was twice as high (P < 0.05) in these animals as compared with L1+L2 (F+A: 0.50±0.13; L1+L2: 0.25±0.09 lambs born per inseminated ewe). In experiment 2, oestrous cycles of 40 ewes were synchronised with two i.m. injections of 7.5 mg Luprostiol. Sev- enty-two hours after the second injection (day 0), progestagen pessaries were inserted in 20 ewes (Group 0), when the ewes were in the follicular phase of the cycle. The remaining 20 ewes received the pessaries on day 11, during the luteal phase of the cycle (Group 11). Animals were hand-mated at least twice by fertile rams 48 h later; the ewes were thereafter exposed to rams in the pen overnight. The percentage of fertility was 90% in both groups. No significant differences between groups were observed for litter size and fecundity. In conclusion, an onset of the synchronisation treatment with progestagen in a program of artificial insemination close to the follicular phase of the oestrous cycle can improve both fertility and the number of lambs born per inseminated ewe. If natural service is used, perhaps the stay of the rams with the ewes for at least 24 h might compensate for the asynchrony between ovulation and mating, leading to similar percentages of fertility, whatever the phase of the cycle when the synchronisation treatment began. Further studies involving a higher number of ani- mals are necessary to confirm these results.

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

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

Ram-induced ovulation to improve artificial insemination efficiency with frozen semen in sheep

Ram effect, defined as shortening of seasonal anestrus in ewes by exposure to the ram, is now well recognized but the underlying mechanisms are still unclear. Little information also exists whether the ram is able to influence the estrus cycle and ovulation. Three experiments were conducted to investigate endocrine response, time of evolation and pregnancy rate of ewes in proestrus, exposed to the ram (treated) or an adult ewe (control). In the first experiment, ewes (n = 20) were treated with fluorgestone acetate pessaries for 12 days and were given eCG and cloprostenol one day before withdrawal of pessaries. On the day after removal of the pessaries ewes in the treated group (n = 10) were exposed to the ram and those in the control group (n = 10) were exposed to an adult ewe. Blood samples were taken for LH assay every 20 min from 2 h before to 24 h after ram exposure. In the second experiment, ewes (n = 120) were induced into proestrus and on the day after removal of the pessaries were exposed to either a ram (n = 60) or a ewe (n = 60) as described above and were laparoscoped 50, 60 or 70 h after pessary withdrawal (n = 20 at each time interval). In the third experiment ewes (n = 90) were induced and exposed to the ram (n = 45) or an adult ewe (n = 45) and inseminated via a laparoscope whit frozen-thawed semen at 50 or 60 h after pessary removal, respectively. Exposure to the ram was followed in 2 h by a marked rise in LH, equivalent to a preovulatory surge in duration and amplitude. It was also followed by concentrated ovulation within 25 to 30 h and by an increased pregnancy rate in exposed ewes (73.3 vs. 53.3%).

Effect of diet quantity and urea supplementation on oocyte and embryo quality in sheep

The objective was to investigate the effects of dietary energy and urea supplementation on oocyte and embryo quality in sheep using in vivo and in vitro experimental models. Sixty-three ewes were fed grass metal at 0.5 or 2.0 times maintenance energy requirements (MER). The diet was supplemented with feed grade urea (U) for half of the ewes on each energy treatment. Ewes were stimulated with 1000 IU eCG and either slaughtered on the day of pessary withdrawal for in vitro embryo production, or mated and slaughtered on Day 5 for embryo recovery. Urea decreased cleavage rate (48.3 vs 39.7%) and consequently blastocyst rate (41.6 vs 36.8%) but the differences were not significant. Oocytes from animals on 2.0 MER had a lower cleavage rate (54.9 vs 36.0%) and blastocyst yield (49.3 vs 31.4%) than those on 0.5 MER. However, there was an interaction between urea and energy for cleavage (P = 0.04) and blastocyst yield (P = 0.03) indicating a variable response to urea in the presence of high energy. This was manifested by a decrease in cleavage rate in the presence of urea and high energy (22%, 8 of 36), and a reduction in blastocyst development (19%, 7 of 36). When blastocyst development rate was expressed as a proportion of cleaved oocytes there was no difference between groups; in addition, there was no difference between groups in terms of blastocyst hatching rate (overall mean 66.1%) or blastocyst cell number on Day 8 (overall mean ± SEM, 138.4 ± 9.0, n=61). The effect of urea on cleavage rate in vivo was more severe. Urea supplementation reduced (P<0.001) the cleavage rate (93 vs 62%). Despite this, the yield of blastocysts was unaffected. Oocytes from ewes on 0.5 MER exhibited a lower (P<0.05) cleavage rate than those on 2.0 MER (66 vs 87%). This effect was also apparent at the blastocyst stage (40.0 vs 50.9%), although the difference was no longer significant. There were no differences in hatching rate (overall mean 70.7%) or blastocyst cell numbers (overall mean ± SEM, 166.3 ± 15.6 n=40). Collectively, these results suggest that both high dietary energy and urea content influence subsequent embryo development in vitro, and the deleterious effects of urea are likely influenced by concomitant energy intake.

Effect of interval between two PGF 2α doses on superovulatory response and embryo recovery in crossbred sheep in tropics

An experiment was conducted to investigate the effects of different superovulatory regimes, including pregnant mare serum gonadotrophin (PMSG) and follicle-stimulating hormone (FSH-P) on steroidogenesis, oestrus, ovulation and premature luteal regression in sheep. Thirty mature Merino ewes were treated in the breeding season with intravaginal progestagen pessaries and 1200 IU PMSG (Group 1), 15 mg FSH-P (Group 2) or a combination of 700 IU PMSG and 11 mg FSH-P (PMSG/FSH-P; Group 3) to induce superovulation. After treatment with FSH-P alone, the incidence of oestrus was lower than after treatment with PMSG alone (6/10 vs. 10/10, P < 0.05) and the incidence of ovulation was lower than after treatment including PMSG (7/10 vs. 20/20, P < 0.05). However, there were no differences in the mean time to onset of oestrus (24.7 ± 2.3 h after pessary withdrawal) or the time to onset of ovulation (48–54 h after pessary withdrawal) between the treatment groups.The total ovarian response (corpora lutea + large follicles) was higher (P < 0.05) following stimulation with PMSG alone (17.1 ± 1.9) than with FSH-P (8.3 ± 12.5). However, the numbers of corpora lutea present on Day 6 (Day 0 = pessary withdrawal) were not affected by the exogenous gonadotrophin treatment (PMSG, 11.4 ± 1.8; FSH-P, 7.2 ± 2.5; PMSG/FSH-P, 11.1 ± 2.1; not significant). The mean pre-ovulatory peripheral plasma peak level of oestradiol-17β (E2) per follicle was higher following treatment with PMSG than with either of the other two regimes (PMSG, 23.2 ± 0.8 pg ml−1; FSHP, 10.8 ± 0.6 pg ml−1; PMSG/FSH-P, 13.9 ± 0.4 pg ml−1; P < 0.05). Moreover, E2 peaked earlier in the ewes treated with PMSG than with FSH-P or PMSG/FSH-P (7.6 ± 2.1 h vs. 33.7 ± 1.5 h and 24.8 ± 0.5 h respectively; P < 0.01). There were no differences detected in the peak levels of luteinizing hormone (LH) (8.6 ± 1.1 ng ml−1) or the mean time to LH peak (32 ± 1.4 h) between the ewes treated with the different exogenous gonadotrophins.Three ewes (one treated with PMSG alone and two with PMSG/FSH) had premature luteal regression by Day 6. In each case, a secondary peak of E2 secretion was observed 36–44 h after pessary withdrawal. These ewes each displayed a second oestrus on Days 5–6 and a mild superovulatory response at this oestrus.It is concluded that in sheep PMSG is highly steroidogenic in comparison with FSH-P. A combination of moderate doses of PMSG and FSH-P is a suitable superovulation regime for Merino ewes as it induces a consistently high superovulatory response without the secretion of excessive E2 levels. Perturbation of the periovulatory endocrine events may result in premature luteal regression.

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

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

Superovulatory response in Corriedale sheep during different months of the breeding season

Ovulatory response in 27 Corriedale ewes (born in 1991) was studied by stimulation with exogenous hormones during the months of July and August, 1994. One vaginal pessary per animal, containing fluorogestone acetate (FGA; 45 mg), was kept in situ for 9 days. Administration of PMSG (1000 IU) 1 day before pessary withdrawal induced oestrus and ovulation in all animals, except two which lost their pessaries. The ovulation rate was higher (7.6) in ewes stimulated in August than in those stimulated in July (5.2) ( P < 0.05). The right ovary tended to carry more corpora lutea than the left ( P > 0.05). We can conclude that short-term progesterone therapy (9 days) is as effective as long-term therapy (14 days) for superovulation in sheep and the right ovary is more active than the left.

Embryo production and endocrine response in ewes superovulated with PMSG, with or without monoclonal anti-PMSG administered at different times

Mature nonlactating Altamurana ewes (n = 168) were synchronized in the seasonal anestrus period with FGA-impregnated intravaginal pessaries for 12 d. In Experiment 1, 48 ewes were divided into a 3 × 4 factorial design for anti-PMSG monoclonal antibody (AP) bioassay test. Concomitant injections of PMSG (1000, 1500, 2000 IU) and AP (0, 1, 2, 3 μl/IU PMSG) were given, and ovarian response was evaluated by laparoscopy. In Experiment 2, 120 ewes were divided into 8 experimental groups (n = 15 per group). The ewes treated with 1000 or 1500 IU PMSG at −24 h from sponge removal were given AP intravenously at 50 h after pessary withdrawal, 12 or 24 h after the onset of estrus, while the controls did not receive AP. Blood samples were collected from ewes (n = 6) treated with 1500 IU PMSG with or without anti-PMSG. Ovarian response and embryo production were evaluated on Day 7 after sponge removal upon laparotomy. It was found that 1 μl AP was effective in neutralizing 1 IU PMSG. No significant differences in serum concentrations of progesterone were observed among the groups of superovulated ewes. Estradiol-17 β levels were reduced following AP treatment 12 h after the onset of estrus. At a lower dosage of superovulatory treatment (1000 IU PMSG), AP injected at 12 or 24 h after the onset of estrus significantly lowered large follicles (P < 0.01) and increased the rate of ovulation (P < 0.05). Moreover, embryo production showed a more than two-fold increase (P < 0.01) of viable embryos following AP injection at 12 or 24 h after the onset of estrus (3.2 to 3.3 vs 1.3, with vs without anti-PMSG). It is concluded that superovulatory treatment with 1000 IU PMSG plus AP administered at a fixed time after the onset of estrus may improve ovarian response and the yield of viable embryos in ewes.

Heterogeneity in ovarian steroid secretion response to treatment with PMSG in ewes during the breeding season and anestrus

To determine the range of responses in ovarian steroid secretion to PMSG treatment, ovarian steroid secretion rates and circulating FSH concentrations were studied in ewes with ovarian autotransplants following treatment with or without PMSG (400 IU) during the breeding season and anestrus. The ewes were treated with progestagen pessaries for 12 d. A prostaglandin analogue (PG) was administered at the time of pessary withdrawal to induce luteolysis (0 h). The PMSG was also administered at 0 h. Jugular and ovarian venous blood samples were taken at 10-min intervals at 2 stages (21 to 27 h and 38 to 42 h post PG). There was no change in peripheral plasma FSH concentrations in control ewes with a change in season. Mean peripheral plasma FSH concentrations decreased in PMSG-treated ewes relative to controls in both the breeding season and seasonal anestrus (P < 0.01). Treatment with PMSG increased the ovarian androstenedione and estradiol-17β secretion rates relative to that of the controls in both the breeding season and seasonal anestrus (P < 0.05). The rate of estradiol-17β secretion in PMSG-treated ewes was lower in seasonal anestrus than in the breeding season (P < 0.05). There was a greater range in ovarian steroid secretion response between PMSG-treated individuals, which was most apparent during seasonal anestrus.

Changes in progesterone secretion following treatment with transforming growth factor (TGF- ) during the follicular phase of the sheep oestrous cycle

The effects of transforming growth factor alpha (TGF-alpha) on ovarian steroid secretion were investigated. Three crossbred ewes synchronized for oestrus with ovarian autotransplants were infused with TGF-alpha (30 micrograms in 12 h) via the ovarian artery for 12 h before withdrawal of progestagen pessary. Three ewes were used as controls. Jugular and ovarian venous blood samples were taken at intervals of 10 min at two stages during the follicular phase (21-27 h and 38-42 h after pessary withdrawal) and every 2 h from 44 to 86 h. Plasma LH and FSH concentrations, and ovarian secretion rates of inhibin, androstenedione, oestradiol and progesterone were determined using radioimmunoassays. LH pulse amplitude increased in ewes treated with TGF-alpha in the early follicular phase (0.92 +/- 0.25 micrograms l-1 in controls versus 3.10 +/- 0.35 micrograms l-1 in TGF-alpha treated ewes; P < 0.05) and remained high in the late follicular phase. Plasma FSH concentrations were high during the follicular phase in ewes treated with TGF-alpha (P < 0.05). The infusion of TGF-alpha had no significant effect on the ovarian rate of secretion of androstenedione and, although the secretion rates of oestradiol and inhibin were consistently lower in TGF-alpha-infused ewes, the differences were not significant. The ratio of secretion of androstenedione to oestradiol was greater during the follicular phase in TGF-alpha-treated ewes (P < 0.05), suggesting that the efficiency of aromatization had been impaired.(ABSTRACT TRUNCATED AT 250 WORDS)