Induction of fertile oestrus in seasonally anoestrous ewes with low doses of Gn-RH

Oestrus, conception and lambing performance were assessed in progesterone-primed seasonally anoestrous ewes induced to ovulate with gonadotrophin-releasing hormone (Gn-RH), which was administered intravenously for 48 h as either injections of 250 ng at 2-h intervals (n = 15) or as a continuous infusion at the rate of 125 ng/h (n = 12) or 250 ng/h (n = 12).In $\text{14}\text{15}$ of the ewes injected with Gn-RH, a preovulatory LH peak was recorded at a mean time interval of 33.9 ± 1.8 h after the start of treatment. All ewes displayed oestrus and all ovulated, with a mean ovulation rate of 1.67 ± 0.13. Eleven ewes were diagnosed as pregnant and subsequently lambed. Following infusion of Gn-RH, preovulatory LH peaks were recorded in $\text{21}\text{24}$ ewes at a mean time of 36.1 ± 2.9 h (125 ng/h) and 34.7 ± 2.0 h (250 ng/h). All but two of the ewes displayed oestrus and $\text{23}\text{24}$ ovulated. The group mean ovulation rates of 1.27 ± 0.14 (125 ng/h) and 1.75 ± 0.22 (250 ng/h) were not significantly different. Eleven of the 22 ewes mated were diagnosed as pregnant and produced live lambs.These results suggest that fertility of Gn-RH-induced ovulations in seasonally anoestrous ewes is comparable to that apparent in ewes ovulating spontaneously during the breeding season.     

Treatment of seasonally anestrous Romney ewes with continuous infusion of low doses of GnRH: effects on estrus, ovulation and plasma progesterone concentration

The aim of this study was to assess the suitability of a GnRH infusion regimen (125 ng/h or 250 ng/h) to induce estrous behaviour, ovulation and normal corpus luteum function in progesterone-primed Romney ewes each month of seasonal anestrus (i.e. September to February inclusive) over two years. None of the progesterone-primed control ewes (i.e. no GnRH treatment; N = 120 observations) ovulated, showed normal corpus luteum function or displayed estrous behaviour at any time during anestrus. Approximately 27 and 50% of the respective 125 ng/h and 250 ng/h GnRH-treated ewes (N = 120 observations per GnRH treatment) ovulated and showed normal luteal function. Of those which ovulated 59.2% and 52.4% in the respective 125 ng/h and 250 ng/h GnRH treatment groups showed estrous behaviour. There was a significant effect of GnRH dose on the median number of ovulations (250 ng/h > 125 ng/h; P<0.01) but no overall difference (when both treatment years and GnRH doses were pooled) in the median number of ovulations per month of anestrus. The frequency of ewes with an ovulation rate >2 was low with only 4/95 treated ewes with more than 2 corpora lutea (CL). Treatment of progesterone-primed ewes with 250 ng/h GnRH increased plasma LH (P<0.01) but not FSH concentrations; a significant increase in LH pulse amplitude (P<0.05) but not LH pulse frequency was observed. The plasma gonadotropin levels in the 125 ng/h GnRH treatment groups were not studied. We suggest that in breeds such as the Romney which have a strict (i.e. 5-6 month) anovulatory interval, the GnRH-infusion technique may be of limited practical use for inducing pregnancies during the non-breeding season.     

Plasma FSH concentrations in seasonally anoestrous ewes induced to ovulate with repeated injections or continuous infusion of GnRH

Plasma FSH concentrations were monitored in 34 seasonally anoestrous ewes in which ovulation was induced by the administration of low doses of GnRH, given as either a series of i.v. injections (75, 125, 250 or 500 ng/2 h) or as a continuous i.v. infusion (125 or 250 ng/h). Fifteen of the animals had been pretreated with progesterone for 14 days. Before the start of GnRH treatment, mean FSH concentrations did not differ between progesterone-pretreated and non-pretreated ewes (23 ± 3.0 and 20 ± 2.0 ng/ml, respectively). In a significant (P < 0.01) proportion of animals mean FSH concentrations were elevated for the first 2 h of GnRH treatment, but thereafter they declined progressively and were significantly (P < 0.001) lower than pretreatment levels over the second 12 h of GnRH treatment. These changes in FSH concentrations were not related to dose of GnRH, the mode of administration or to progesterone priming. These results demonstrate that the pattern of FSH secretion associated with GnRH-induced ovulation in the seasonally anoestrous ewe is similar to that observed from the time of luteal regression in the naturally cycling ewe. In addition, although pretreatment with progesterone has a marked effect on subsequent luteal function, this is not mediated through changes in plasma FSH concentrations.     

Suppression of plasma FSH concentrations with bovine follicular fluid blocks ovulation in GnRH-treated seasonally anoestrous ewes

The specific requirement for FSH in the final stages of preovulatory follicle development was assessed in seasonally anoestrous ewes given 2-h injections of GnRH (250 ng/injection), with (N = 10) or without (N = 10) concurrent treatment with bovine follicular fluid (bFF: 2 ml given i.v. at 8-h intervals). Treatment with bFF significantly (P less than 0.01) suppressed plasma FSH concentrations, but, at least for the first 30 h of treatment, did not influence the magnitude of GnRH-induced LH episodes (mean max. conc. 3.00 +/- 0.39 and 3.63 +/- 0.51 ng/ml for bFF-treated and control ewes, respectively). Of 10 animals treated with GnRH for 72 h, 5/5 control ewes showed oestrus and ovulated whereas 0/5 bFF-treated ewes showed oestrus or ovulated in response to GnRH treatment. There was, however, a transient (13.2 +/- 1.0 h) increase in plasma LH concentrations in the ewes given bFF (mean max. conc. 4.64 +/- 1.57 ng/ml), which was coincident with the preovulatory LH surge recorded in animals given GnRH alone. In 10 GnRH-treated ewes slaughtered after 32 h of treatment, the mean diameter of the largest antral follicle was significantly (P less than 0.001) greater in control ewes (5.92 +/- 0.17 mm) than in animals that were also given bFF (3.94 +/- 0.14 mm). In addition, the incidence of atresia in the 3 largest antral follicles present at this time was greater in bFF-treated ewes. These results show that, when plasma FSH concentrations are suppressed by administration of bFF, although the magnitude of GnRH-induced LH episodes is unchanged, preovulatory follicular development is impaired and ovulation does not occur.(ABSTRACT TRUNCATED AT 250 WORDS)     

Response of seasonally anoestrous ewes to six-hour periods of GnRH infusion administered on six consecutive days

Twelve seasonally anoestrous Clun Forest ewes were infused i.v. with either 500 or 1000 ng GnRH/h for 6 h on each of six consecutive days in early or mid-anoestrus. Plasma LH concentrations were elevated during each GnRH infusion but returned to pretreatment levels when infusion ceased. The response to the first infusion was significantly greater (p<0.001) than that to subsequent infusions. In addition, both a GnRH dose and a seasonal influence were evident in the LH response, but these failed to reach statistical significance. Although 7 12 ewes ovulated, only two produced functionally normal corpora lutea.     

Steroid production and hCG binding by ram-induced ovarian follicles in seasonally anoestrous ewes

The introduction of rams to a group of previously isolated anoestrous ewes has been shown to stimulate ovarian follicular development and ovulation. The present experiment was carried out to determine the ability of follicles arising from this ram stimulus to produce steroids and bind hCG. Seasonally anoestrous Southdown ewes were exposed to rams for 24 h, 40 h, 3 days, 10 days or 20 days before ovariectomy. Steroid production and the concentration of hCG binding sites in follicles dissected from the ovaries were measured in vitro. The presence of a ram caused ovulation and enhanced oestradiol production by follicles, but had little effect on total androgen production or the number of hCG binding sites present in the follicles when compared to follicles from anoestrous ewes. The oestradiol concentrations in large follicles were not as high as in preovulatory follicles from cyclic ewes reported in other studies. Follicles continued to develop through the ram contact period and when incubated after 40 h and 10 days of ram contact produced high levels of progesterone, indicating partial luteinization, although the corpora lutea (CL) resulting from the induced ovulations regressed prematurely. We suggest that the lack of hCG binding sites in ram-induced follicles may be the cause of poor luteinization and suboptimal development of luteal tissue after induced ovulation in ewes during seasonal anoestrus.     

The effect of exposure to oestrous ewes on rams' sexual behaviour, plasma testosterone concentration and ability to stimulate ovulation in seasonally anoestrous ewes

Ruminants eat a variety of foods from different locations in the environment. While water, cover, social interactions, and predators are all likely to influence choice of foraging location, differences in macronutrient content among forages may also cause ruminants to forage in different locations even during a meal. We hypothesized that lambs forage at locations containing foods that complement their basal diet and meet their nutritional needs. Based on this hypothesis, we predicted that lambs (n=12) fed a basal diet low in protein and high in energy would forage where a high-protein food (Food P) was located, and that lambs (n=12) fed a basal diet low in energy and high in protein would forage where a high-energy food (Food E) was located. Food P was a ground mixture of blood meal (50%), grape pomace (30%), and alfalfa (20%) that contained 47% crude protein (CP) and 2.211 Mcal/kg digestible energy (DE). Food E was a ground mixture of cornstarch (50%), grape pomace (30%), and rolled barley (20%) that contained 6% CP and 3.07 Mcal/kg DE. Food P provided 212 g CP/Mcal DE, whereas Food E provided 20 g CP/Mcal DE. Lambs growing at a moderate rate require 179 g CP and 3.95 Mcal DE. During Trial 1, we determined if lambs foraged to correct a nutrient imbalance, and if they preferred a variety of foods (Foods P and E) to only one food at a location (Food P or E). During Trial 2, we determined if nutrient-imbalanced lambs foraged in the location with the food that corrected the imbalance when the location of the foods changed daily. During Trial 3, lambs were offered familiar foods (Foods P and E) at the location furthest - and novel foods (wheat and soybean meal) at the location nearest - the shelter of their pen. During all three trials, lambs foraged most at the location with the food that contained the highest concentration of the macronutrient lacking in their basal diet, but they always ate some of both foods. Lambs did not feed exclusively at the location with a variety of foods (P and E). Rather, they fed at the location nearest the shelter that contained the macronutrient lacking in their diet. As availability of the food with the needed macronutrient declined in one location, lambs moved to the nearest location that had food with the needed macronutrient. When food that complemented their basal diet was moved to a different location, lambs foraged in the new location. Collectively, these results show that lambs challenged by imbalances in energy or protein selected foods and foraging locations that complemented the nutrient content of their macronutrient imbalanced basal diets.     

Ovarian capillary blood flow in seasonally anoestrous ewes induced to ovulate by treatment with GnRH

The microsphere technique was used to obtain estimates of ovarian capillary blood flow near ovulation, in 8 seasonally anoestrous ewes, which were induced to ovulate by GnRH therapy. Plasma progesterone concentrations were monitored in jugular blood sampled between Days 4 and 7 after the onset of the preovulatory LH surge. The ewes were then slaughtered. Three of the ewes were treated with a single injection of 20 mg progesterone before GnRH therapy. In these ewes and 1 other, plasma progesterone values increased after ovulation and reached 1.0 ng/ml on Day 7 following the preovulatory LH surge (normal, functional CL), whilst in the other 4 ewes progesterone concentrations increased initially then declined to 0.5 ng/ml by Day 7 (abnormal CL). In the ewes exhibiting normal luteal function, the mean ovarian capillary blood flow was significantly greater (P less than 0.01) than that for ewes having abnormal luteal function. Irrespective of the type of CL produced, capillary blood flow was significantly greater (P less than 0.05) in ovulatory ovaries than in non-ovulatory ovaries. These findings indicate that the rate of capillary blood flow in ovaries near ovulation may be a critical factor in normal development and maturation of preovulatory follicles and function of subsequently formed CL.     

Exogenous GnRH induces ovulation in seasonally anoestrous lactating goats (Capra hircus)

A specific sheep LH radioimmunoassay was validated for the measurement of goat LH, and used to monitor luteal-phase LH episodes and the preavulatory LH surge in progestagen sponge-synchronized cycling goats. No luteal-phase LH episodes were detected during 12 h of frequent (15-min) blood sampling in 2 goats. A preovulatory LH surge was recorded in 5/5 goats, with a mean amplitude of 45.4 +/- 7.2 ng/ml and a mean time of onset of 38.4 +/- 1.2 h after removal of a progestagen-impregnated sponge. In anoestrous goats, single i.v. injections of 1000 and 2000 ng GnRH induced LH episodes with a mean amplitude of 2.04 +/- 0.11 and 3.67 +/- 0.06 ng/ml respectively, but injections of 250 or 500 ng did not consistently elevate LH concentrations. Progestagen-primed, seasonally anoestrous lactating goats were treated with repeated injections of 1500 ng GnRH (every 2 h for 52 or 78 h) in May 1985 or 1986. All 10 had kidded in March of the same year, and were consequently at peak lactation at the time of GnRH treatment. A preovulatory LH surge was detected in 9 goats with a mean time of onset of 59.5 +/- 2.9 h (1985) or 39.6 +/- 3.3 h (1986) after vaginal sponge removal. All animals displayed oestrus and ovulated, and 9 of the goats were mated: in 5 of these animals pregnancies were successfully carried to term. The results show episodic LH release in response to GnRH and indicate that ovulation can be induced in seasonally anoestrous goats, even at peak lactation, and normal pregnancies may result.     

Pulsatile GnRH administration stimulates the number of pituitary GnRH receptors in seasonally anoestrous ewes

Twenty seasonally anoestrous ewes were pretreated with progesterone for 4 days and divided into four equal groups. Ewes in Group 1 received no GnRH treatment and were slaughtered immediately after progesterone removal. Ewes in Groups 2, 3 and 4 received i.v. injections of 250 ng GnRH every 2 h for 36 h starting at the time of progesterone removal. Ewes in Group 2 were slaughtered immediately after the 36 h GnRH pulsing, while ewes in Groups 3 and 4 were given a bolus injection of 125 micrograms GnRH at this time and were slaughtered 2 and 10 h after the bolus injection, respectively. Blood samples were collected every 30 min from ewes in Group 4 only, from 4 h before the start of GnRH treatment until 10 h after the bolus injection. Pulsing with GnRH resulted in episodic release of LH, and the bolus injection of GnRH was immediately followed by a preovulatory type LH surge in those ewes in which an endogenous surge had no already begun. The pituitary GnRH receptor numbers were significantly higher for the ewes in Group 2 than for any of the other treatment groups, while there was no significant difference in the receptor numbers between Groups 1, 3 and 4. The results suggest an up-regulation of GnRH receptors resulting from pulsatile GnRH therapy.     

Induction of estrus and superovulation in seasonally anestrous ewes

The induction of estrus in 17 previously cycling nulliparous ewes, 9 to 10 months of age, was attempted with Medroxyprogesterone acetate (MAP) pessaries during the early anestrous period (March-April). Ewes were verified to be anestrous by the lack of estrous behavior in the presence of a vasectomized ram and by a radioimmunoassay for serum progesterone in two samples taken 7 days apart showing less than 1 ng/ml serum progesterone. Superovulation was attempted with injections of either FSH or FSH + LH. MAP vaginal pessaries remained in place for a period of 12 days and FSH was administered to all ewes (IM) at 12 hr intervals over a 3 day period; 5 mg was injected twice on day 11 after pessary insertion, followed by 4 and 3 mg injections twice daily on each succeeding day, for a total of 24 mg per ewe. Nine ewes were given 25 mg LH (IV) within 8 hrs after the onset of behavioral estrus in addition to FSH. Ewes were hand-mated to several rams at 12 hr intervals throughout the estrus period. Ovulation and fertilization rates were recorded for each ewe following midline laparotomy and embryo collection. All ewes were in estrus between 36 and 48 hrs after removal of the MAP pessaries. In ewes injected with FSH only, 8 of 8 ovulated with a mean ovulation rate of 6.0 +/- 4.4 and a fertilization rate of 70%. Nine of 9 ewes receiving both FSH + LH ovulated with a mean ovulation rate of 13.9 +/- 13.1 and a fertilization rate of 72%. Statistical analysis by Students t-test resulted in differences in number of ova recovered (P<.05) between FSH only and FSH + LH treated ewes and a trend towards increased ovulation rate in FSH + LH treated ewes. These results show that early seasonally anestrous ewes can be successfully induced and synchronized for estrus with MAP pessaries and the number of ova recovered is increased with the inclusion of LH in the superovulation regime.     

Induction of pulsatile LH release, FSH release and ovulation in post-partum acyclic beef cows by repeated small doses of Gn-RH

Five acyclic spring-calving beef cows (20-40 days post partum) were bled every 15 min for 60 h and thereafter every 6 h for 5 days. Gn-RH (5 micrograms) was injected every 2 h for 48 h, starting 12 h after sampling began. Pulsatile patterns of LH release occurred synchronously in response to injection and 4 of the 5 treated animals subsequently ovulated and completed at least one full ovarian cycle. Four of 6 similar control cows were bled every 10 min for 8-h periods at equivalent times post partum. Pulses of LH were seen after approximately Day 25 post partum with a mean pulse frequency of 0.5 per h. There was little evidence of a discrete pulsatile mode of FSH release in any of the treated or control cows. The time to the first significant progesterone rise in the 4 treated and ovulating cows (34.5 +/- 5.6 days post partum) was significantly shorter (P less than 0.05) than in the 6 control animals (66.3 +/- 11.4 days).     

Ovarian steroid involvement in endogenous opioid modulation of LH secretion in seasonally anoestrous mature ewes

In June, 16 mature ewes were ovariectomized and allocated to four groups: 1, saline; 2, naloxone; 3, progesterone implant plus naloxone; 4, oestrogen implant plus naloxone. Steroids were implanted at the time of ovariectomy. At 5 days after ovariectomy, the animals were intravenously infused with saline for 8 h and naloxone (50 mg/h) in saline for 8 h the following day. Three intact ewes were given naloxone in a similar way. During infusions and for 8 h on the day after naloxone, jugular venous blood samples were taken every 15 min and assayed for LH. Naloxone resulted in significant increases in mean LH concentration (P less than 0.01), LH episode frequency and episode height (P less than 0.05) in Group 3 ewes, but was without effect in any other group. These results provide evidence that the progesterone status of the ewe affects its response to naloxone, that progesterone negative feedback on LH release may be mediated by an opioid system, and that increased oestradiol negative feedback during seasonal anoestrus is unlikely to work via increased opioid inhibition of LH.     

Importance of behavioural stimuli in ram-induced ovulation in seasonally anovular Corriedale ewes

Three groups of about 65 seasonally anovular Corriedale ewes were penned and exposed to rams on November 4, 1989. A fourth group (n=64) was maintained in a 3-ha paddock and also exposed to rams on the same date. The sexual behaviour exhibited by rams in contact with penned ewes was recorded during nine simultaneous observation sessions lasting 2 h each.

Five days after ram introduction, incidence of ovulation and ovulation rate were higher (P < 0.01) in adult than in 1.5-year-old ewes. Among penned adult ewes, a greater (P < 0.05) incidence of ovulation was recorded in ewes maintained in contact with rams and oestrous ewes (95.8%) than in ewes exposed to either rams with or without recent experience with oestrous ewes (68.7 and 57.1%, respectively). The fourth group, exposed to rams in field conditions, exhibited a lower (P < 0.05) incidence of ovulation (31.9%) than any other group. No differences among treatments were detected in 1.5-year-old ewes. The incidence of ovulation was associated (r=0.99, P<0.05) with the sexual behaviour displayed by the rams.

It was concluded that the sexual behaviour of the rams plays an important role in ram-induced ovulation and that the continuous presence of oestrous ewes is necessary for the maximum efficacy of the technique in Corriedale ewes.     

Induction of ovulation in chronically hypophysectomized Booroola ewes

Booroola Merino ewes, with (F+; N = 17) and without (++; N = 13) a copy of the fecundity gene were hypophysectomized and 6 weeks later were given an i.m. injection of PMSG (high, medium or low dose) followed by hCG. The induced ovulation rates were observed laparoscopically. Ovulation rates were significantly higher (P less than 0.01) in Booroola F+ ewes than in ++ ewes (8.00 +/- 1.66 s.e.m. vs 3.62 +/- 1.10 respectively). This suggests that the high fecundity of the Booroola ewe may be due primarily to ovarian rather than pituitary factors.     

Progesterone exposure of seasonally anoestrous ewes alters the expression of angiogenic growth factors in preovulatory follicles

Small-dose, multiple injections of GnRH given to seasonally anoestrous ewes induce final stages of the preovulatory follicle development, but result in an high incidence of defective CL unless animals are primed with progesterone, which completely eliminates luteal dysfunction. Progesterone priming upregulates luteal vascularization; however, its effect on follicular angiogenesis is poorly understood. This study tested the hypothesis that progesterone priming of seasonally anoestrous ewes treated with dose multiple injections of GnRH eliminates defective luteal function by altering the expression of vascular endothelial growth factor (VEGF), VEGF receptor-2, angiopoietin (ANG)-1, ANG-2, and TIE-2 during early and late preovulatory follicle development. Ten seasonally anoestrous ewes were given 20 mg of progesterone im 3 days before the start of GnRH treatment; 10 other animals served as controls. Intravenous injections of 500 ng GnRH were given to all animals every 2 hours for 28 hours, followed at 30 hours with a 300-μg GnRH bolus injection to synchronize the preovulatory LH surge. Ovaries were collected at 24 and 46 hours after the start of GnRH treatment. Small (2–2.5 mm) and large (>2.5 mm) follicles were analyzed for protein and mRNA expression of the angiogenic factors using immunohistochemistry and in situ hybridization assays. Progesterone priming did not have an influence on angiogenic factor levels in small follicles. However, progesterone-primed animals showed significantly (P ≤ 0.05) higher levels of VEGF, VEGFR-2, ANG-1, and ANG-2 in large follicles compared with nonprimed ones. These data suggest that progesterone priming alters the expression of angiogenic factors in large preovulatory follicles, ensuring adequate luteal development and function.     

Effects of recent sexual experience and melatonin treatment of rams on plasma testosterone concentration, sexual behaviour and ability to induce ovulation in seasonally anoestrous ewes

The aim of this study was to determine whether advancing the seasonal changes associated with rams by treatment with exogenous melatonin and allowing the rams previous sexual experience would increase the proportion of anoestrous ewes ovulating in early July. North Country Mule ewes (n = 225) were grouped by live body weight and body condition score and allocated randomly to the following treatments: (i) isolated from rams (control; n = 25); (ii) introduced to rams (treatment 2); (iii) introduced to rams that had mated with ewes during the previous 2 days (treatment 3); (iv) introduced to rams implanted with melatonin (treatment 4); and (v) introduced to rams that were implanted with melatonin and had mated with ewes during the previous 2 days (treatment 5). Treatments 2-5 were replicated (2 x 25 ewes) and two rams were introduced to each replicate group. Introductions began on 4 July and were completed by 11 July. The rams were withdrawn from the ewes after 8 days. Melatonin was administered as a subcutaneous implant (Regulin((R))) on 22 May and again on 20 June. Blood samples were taken from all rams to determine plasma melatonin and testosterone concentrations (19 samples in 6 h). The behaviour of the sheep was videotaped continuously during the first 3 h after the ram was introduced. Ovulation was detected by an increase in plasma progesterone concentrations from < 0.5 ng ml(-1) to > 0.5 ng ml(-1). Mean +/- SE plasma melatonin concentrations were 649.7 +/- 281.4 and 18.3 +/- 2.4 pg ml(-1) in rams with and without melatonin implants, respectively (P < 0.001). Melatonin implants also increased plasma testosterone concentrations from 4.30 +/- 1.88 to 10.10 +/- 1.10 ng ml(-1) (P < 0.01), the libido of the rams and the proportion of ewes that ovulated in response to the rams (43 and 56% (treatments 4 and 5) versus 24% (treatments 2 and 3)). In conclusion, implanting rams with melatonin before introducing them to seasonally anoestrous ewes increases the proportion of ewes that ovulate in response to introduction of a ram, but previous sexual experience of rams appears to have little or no effect.