Effect of time and dose of recombinant follicle stimulating hormone agonist on the superovulatory response of sheep

The objective of this study was to determine the superovulatory potential of a single-chain analog of human FSH (Fcα) when administered to ewes either 3 days before, or coincident with, simulated luteolysis (pessary removal [PR]). A total of 40 animals were randomly assigned to receive Fcα at doses of 0.62, 1.25, or 2.5 IU/kg of body weight (bwt) 3 days before PR or 0.31, 0.62, 1.25, or 2.5 IU/kg of bwt at PR. Control ewes received protein without FSH activity. Blood samples were collected during the periovulatory period and ovarian tissue was collected 11 days after PR. Ovulation rate did not differ from the control group in ewes receiving the smallest doses of Fcα (0.31 and 0.62 IU/kg). However, a significant superovulatory response was noted in sheep receiving Fcα at doses of 1.25 and 2.5 IU/kg and this response was comparable in animals receiving the largest dose levels of Fcα at, or 3 days before, PR. The interval between PR and the LH surge was significantly extended and the LH surges were less synchronous in animals receiving Fcα at PR when compared with animals receiving the potent FSH agonist 3 days before PR. Taken together, these data indicate that the human single-chain gonadotropin with FSH activity promotes superovulation in ewe lambs in the breeding season. A single injection of the recombinant gonadotropin 3 days before luteolysis synchronizes the LH surge. The use of the single-chain analog of FSH in assisted reproduction for domestic animals is likely to be of practical significance as an alternative to conventional gonadotropins in superovulation protocols in livestock species.     

Effects of naloxone,morphine and methionine enkephalin on serum prolactin,luteinizing hormone,follicle stimulating hormone,thyroid stimulating hormone and growth hormone

The response of 5 anterior pituitary hormones to single injections of naloxone, morphine and metenkephalin administration was measured in male rats. Morphine and met-enkephalin significantly increased serum prolactin and GH concentrations, and significantly decreased serum LH and TSH concentrations. Naloxone reduced serum prolactin and GH concentrations, increased serum LH and FSH, but had little effect on serum TSH concentrations. Concurrent injections of naloxone with morphine or met-enkephalin reduced the response to each of the drugs given separtely. These results suggest that endogenous morphinomimetic substances may participate in regulating secretion of anterior pituitary hormones.     

Effects of recombinant human follicle stimulating hormone on follicle development and ovulation in the mare

The only gonadotrophin preparation shown to stimulate commercially useful multiple ovulation in mares is equine pituitary extract (EPE); even then, the low and inconsistent ovulatory response has been ascribed to the variable, but high, LH content. This study investigated the effects of an LH-free FSH preparation, recombinant human follicle stimulating hormone (rhFSH), on follicle development, ovulation and embryo production in mares. Five mares were treated twice-daily with 450 i.u. rhFSH starting on day 6 after ovulation, coincident with PGF(2alpha) analogue administration; five control mares were treated similarly but with saline instead of rhFSH. The response was monitored by daily scanning of the mares' ovaries and assay of systemic oestradiol-17beta and progesterone concentrations. When the dominant follicle(s) exceeded 35 mm, ovulation was induced with human chorionic gonadotrophin; embryos were recovered on day 7 after ovulation. After an untreated oestrous cycle to 'wash-out' the rhFSH, the groups were crossed-over and treated twice-daily with 900 i.u. rhFSH, or saline. At the onset of treatment, the largest follicle was <25 mm in all mares, and mares destined for rhFSH treatment had at least as many 10-25 mm follicles as controls. However, neither dose of rhFSH altered the number of days before the dominant follicle(s) reached 35 mm, the number of follicles of any size class (10-25, 25-35, >3 mm) at ovulation induction, the pre- or post-ovulatory oestradiol-17beta or progesterone concentrations, the number of ovulations or the embryo yield. It is concluded that rhFSH, at the doses used, is insufficient to stimulate multiple follicle development in mares.     

Effects of follicle stimulating hormone and purines on rat oocyte maturation

The presented data demonstrate a dose-dependent inhibition of spontaneous meiosis of cumulus-enclosed rat oocytes by guanosine, hypoxanthine, and adenosine. The inhibition by adenosine was transient whereas guanosine and hypoxanthine exerted a persistent effect over 24 h of incubation. The order of potency of the substances was guanosine greater than hypoxanthine greater than adenosine and the inhibition was reversible. The inhibitory effect was reduced when the cumulus cells around the oocyte were removed. The inhibition during the first 12 h of incubation was potentiated by FSH. However, at 24 h of incubation FSH partially overcame the inhibitory effect by hypoxanthine but did not influence the inhibitory effect by guanosine. Also 8BrcAMP potentiated the inhibitory effect observed by guanosine, hypoxanthine, and adenosine, suggesting that the potentiating effect of FSH was mediated via cAMP. Our data demonstrate that adenosine, hypoxanthine, and guanosine synergized with FSH in inhibiting spontaneous rat meiosis, as previously shown in mouse. FSH could partially overcome the inhibitory effect exerted by hypoxanthine but did not counteract the inhibitory effect of guanosine.     

Superovulating cows with follicle stimulating hormone and pregnant mare's serum gonadotrophin

A total of 511 embryos was recovered non-surgically from nearly 100 superovulated or untreated donors. Superovulation with FSH-LH resulted in more corpora lutea, recovered ova, and pregnancies (P<.01) than superovulation with PMSG. No differences were observed in numbers of ovulations, embryos recovered, or pregnancies per donor when prostaglandin F_2α was given to donors 2 versus 3 days following gonadotrophin treatment.Pregnancy rates of 12, 31, 58, and 63% were obtained from groups of embryos classified morphologically as poor, fair, good, and excellent (P<.05). Morphologically normal embryos collected and transferred at 5 to 6 days of gestation resulted in more (P<.05) fetuses (75%) than morphologically normal embryos at 8 to 9 days of gestation (56%), but neither was significantly different from morphologically normal embryos at 6.5 to 7.5 days of gestation (61%). There was no difference (P>.05) between pregnancy rates when retarded embryos from untreated donors (12%) were compared to retarded embryos from superovulated donors (22%). However, a higher proportion of morphologically normal embryos from untreated donors developed into fetuses (71%) than did morphologically normal embryos from superovulated donors (59%, P<.05).     

Continuous subcutaneous infusion of follicle stimulating hormone as a method of superovulating dairy cows

Twenty-eight Holstein cows were randomly assigned to two treatments. Group I received 50 mg of follicle stimulating hormone-pituitary (FSH-P) intramuscularly (i.m.) in declining doses in 5 d. Group II received 49 mg of FSH-P in 7 d via constant-infusion, subcutaneously (s.c.) implanted osmotic pumps. Cows received 25 mg prostaglandin F_2α 48 h after initiation of treatment and were inseminated at estrus. Intramuscular injections resulted in higher numbers of corpora lutea (CL) and embryos than the s.c. infusion. These differences were largely attributed to differences in the doses of FSH-P administered in the two groups. It was concluded that continuously infusing FSH-P s.c. via osmotic pumps can be used as a method of superovulating dairy cows.     

Testosterone, luteinizing hormone, follicle stimulating hormone, and prolactin response to unilateral castration in prepubertal Holstein bulls

Hemicastration of Holstein bulls at 3 months of age resulted in increased (P<0.005) testicular weitht and testis sperm cell content at 330 days after treatment, but did not alter sperm cell concentration in the remaining hypertrophied testis. Radioimmuroassay of blood hormones at 1, 6, 12, and 24 weeks after treatment revealed that unilateral castration did not alter (P>0.1) basal levels or GnRH response profiles of either LH or testosterone compared to intact bulls. Hemicastration caused FSH to be elevated (P<0.01) compared to intact bulls at all sampling periods in both unstimulated and GnRH stimulated bulls. Prolactin varied with season and was greater (P<0.001) in hemicastrated bulls than in intact bulls at 1 and 6 weeks after treatment. Results indicate that unilateral castration at 3 months of age caused testicular hypertrophy of both steroidogenic and gametogenic function and this phenomena may be triggered by increased FSH or prolactin secretion, or both. Further, results indicate different testicular regulation mechanisms exist for pituitary LH and FSH release in bulls.     

Preovulatory evaluation of the superovulatory response in donor cattle

Dairy cows and heifers (n = 134) were induced to superovulate with exogenous gonadotrophins. In 103 animals, peripheral plasma concentrations of progesterone (P4) and luteinizing hormone (LH) were measured during the preovulatory period. On the basis of these measurements, normal and deviating profiles of P4 and LH were defined. A high degree of correlation existed between the normality of the two profiles; when the P4 profile was normal, the probability for the LH profile also to be normal was greater than 10:1. This relationship was utilized to evaluate donors based on four preovulatory measurements of P4. When used on 31 animals used for collection of eggs, a superior superovulatory response was encountered in animals with normal vs deviating P4 profiles (eggs recovered: 7.2 +/- 1.1 vs 0.5 +/- 0.3, P < 0.001; transferable embryos: 4.4 +/- 0.9 vs 0.3 +/- 0.2, P < 0.01). It is concluded that evaluation of donors by measurements of progesterone in plasma at four preovulatory sampling points allows for the early exclusion of donors with inferior embryo yield.     

Effects of suckling frequency and breed on productive performance,follicular dynamics and postpartum interval in beef cows

One experiment was conducted to determine the influence of suckling frequency (restricted suckling (RS) versus ad libitum suckling (AS)) and breed (Brown Swiss versus Pirenaica) on productive performance, follicular dynamics and postpartum cyclicity resumption in beef cows. Twenty-four spring-calving cows (12 Brown Swiss and 12 Pirenaica) were used. After calving cows were assigned, within breed, to the two suckling frequencies (RS versus AS). During the postpartum period, cows were fed 100% of the daily energy estimated requirements. The study was conducted from calving to either day 90 postpartum or to detection of the second ovulation whenever it came later. The pattern of follicular development was monitored daily by transrectal ultrasonography, and blood samples were collected three times a week to analyse progesterone concentrations by RIA. Body condition score at calving was 2.5 (from 1 to 5). Productive parameters were not significantly affected by suckling frequency. Pirenaica cows gained more weight (P<0.001), while Brown Swiss cows had higher milk yield (P<0.001) and thus their calves grew faster (P<0.05). In the present moderate conditions of nutrition, either suckling frequency or breed did not affect follicular dynamics (on average there were 6.5 follicles in emergence, growth rate of dominant follicle (DF) was 1.3mm per day and maximum diameter of dominant follicle was 13.9mm). There were gradual increases in maximum diameter and growth rate of dominant follicle related to the proximity of the ovulatory waves, and they were defined by third order polynomial equations. Postpartum anoestrous interval (PPI) was markedly affected by the interaction between suckling frequency and breed (P<0.001): ad libitum suckling prolonged significantly the interval from calving to first ovulation in Brown Swiss cows (31.8 day versus 82.7 day, in RS and AS groups, respectively, P<0.05), but not in Pirenaica cows (43.5 day versus 32.6 day, in RS and AS groups, respectively, not statistically significant). It is concluded that in moderate pre- and postpartum nutritional conditions Brown Swiss cows were more susceptible to the suppressor effect of ad libitum suckling on postpartum anoestrous interval than Pirenaica cows, although this breed difference did not seem to be mediated by differences in energy balance or in ovarian follicular dynamics.     

Studies on the follicle stimulating hormone releasing hormone by radioimmunoassays and by bioassays

An entity (in fractions), separated from the luteinizing hormone-releasing hormone (LHRH), <Glu-OH, and N-Ac-Asp-OH, which released both FSH and LH appeared to show immunoreactivity in the RIA for LHRH. This entity was destroyed by trypsin, but did not yield LHRH, under conditions which (1) converted a synthetic model, [Arg-Lys-Gln¹]-LHRH, of a pro-LHRH to LHRH; (2) did not destroy LHRH. This entity may not be a pro-LHRH, but may be the follicle stimulating hormone-releasing hormone (FSHRH) on the basis of all these data. A second immunoreactive entity had negligible, if any, releasing activity for FSH and LH, and did not yield LHRH on trypsin digestion.     

The effect of PMSG-priming on subsequent superovulatory response in dairy cows

Superovulation was induced in 56 dairy cows to evaluate the effect of two different regimens using pregnant mare serum gonadotropin (PMSG). Thirty-two cows (controls) were superovulated between Days 9 and 12 of the estrous cycle with a single dose of PMSG (2 800 IU), while remaining 24 cows (PMSG-primed) received 200 IU of PMSG on Day 4 of the estrous cycle and subsequently a single dose of PMSG (2 800 IU) between Days 8 and 12. The cows in both treatments were each given 0,5 mg of cloprostenol at 48 h after the superovulatory PMSG treatment. They were then artifically inseminated twice, 48 h and 72 h later. Embryos were recovered at sloughter between Days 2 and 5 of the cycle and morphologically evaluated. The number of corpora lutea (CL) in the ovaries of the cows was recorded. The mean number of CL (7.2 vs 17.8) was significantly higher (P 0.01) for PMSG-primed cows. The percentage of recovered ova (60.5 vs 70.2 %) and good embryos (79.3 vs 70.7%) were not significantly different between groups. The percentage of fertilized ova (91.4 vs 83.8%) was significantly (P 0.025) greater for the controls. Results of the study indicate that PMSG-priming increased the ovulation rate in the cows superovulated with PMSG.     

Effects of unilateral castration on serum luteinizing hormone, follicle stimulating hormone, and testosterone concentrations in one-, two-, and three-year-old stallions

The endocrine control of compensatory hypertrophy was investigated in 12 Morgan stallions, four each at one, two and three years of age. Half were assigned to be unilaterally castrated (UC) in January and half to remain intact (IN). Nine blood samples were taken from each stallion at half-hour intervals 30, 90, and 150 d after unilateral castration for radioimmunoassay of serum concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), and testosterone. Mean serum LH concentration was greater (P<0.06) in UC than IN stallions; however, the difference was greatest at 30 d and least at 150 d. Serum LH was greater (P<0.01) in two- and three-year-olds than in one-year-olds. The mean log(10) for serum FSH concentration was greater (P<0.06) in UC than IN stallions. Mean serum testosterone concentrations were similar in UC and IN stallions for all sample days, suggesting that the single testes of the UC stallions produced as much testosterone as the two testes of the IN stallions. Two- and three-year-old stallions had greater (P<0.01) serum testosterone than one-year-old stallions. Unilateral castration of stallions was associated with a significant increase in serum LH and FSH concentrations and, perhaps, higher intratesticular testosterone, which may explain, in part, the compensatory hypertrophy noted in the remaining testis.     

Effects of GnRH treatment on superovulatory responses of dairy cows

Twenty-eight Holstein cows were randomly divided into two groups. Group received 50 mg of FSH-P intramuscularly in declining doses for five days. Cows in group II received the same treatment as those in Group I but were also administered 250 ug of GnRH on the day of expected estrus. Neither the mean number of corpora lutea palpated nor the mean number of embryos recovered nonsurgically was different (P0.05) between treatments. However, administration of GnRH resulted in a higher fertilization rate and recovery of more developing embryos.     

Plasma concentrations of luteinizing hormone and progesterone during superovulation of dairy cows using follicle stimulating hormone or pregnant mare serum gonadotrophin

Eighteen lactating Holstein cows were randomly divided into three groups of equal size. Six cows were not superovulated; the remaining cows were superovulated using either FSH-P or PMSG beginning on Day 12 of the estrous cycle (day of ovulation = Day 0). Animals treated with FSH-P were injected intramuscularly (i.m.) with 4 mg FSH-P every 12 h for 5 d. PMSG was administered i.m. as a single injection of 2350 IU. Cloprostenol (PG, 500 ug) was injected i.m. 56 and 72 h after commencement of treatment and at the same time in the cycle of controls. All cows were inseminated 56, 68 and 80 h after the first PG injection. Blood samples (5 ml) were collected daily and every 15 min for a period of 9 h on Days -1, 0, 2, 8 and 10, with continuous blood sampling at 15-min intervals during Days 3 to 6. Ovulation rate was 27.7 +/- 8.22 in animals treated with PMSG, and 8.0 +/- 3.2 embryos per donor were recovered. In the FSH group, ovulation rate was 8.3 +/- 1.48 and 3.0 +/- 1.1 embryos per donor were recovered. Progesterone concentrations were similar in all three groups until the onset of the LH surge, when progesterone concentrations were greater (P<0.05) in animals of the PMSG group. After the preovulatory LH surge, concentrations of progesterone started increasing earlier (44 h) in cows treated with PMSG, followed by FSH-treated cows (76 h) and controls (99 h). The LH surge occurred earlier (P<0.05) in PMSG-treated cows (37 h after first PG treatment), than in animals treated with FSH-P (52 h) or controls (82 h). In animals treated with FSH-P, the magnitude of the preovulatory LH surge (24.2 +/- 1.02 ng/ml) was higher (P<0.05) than in the other two groups (PMSG = 17.1 +/- 2.04 ng/ml; control, 16.7 +/- 1.24 ng/ml). Superovulation with FSH-P or PMSG did not affect either mean basal LH concentration, frequency or amplitude of LH pulses during Days -1, 0, 2, 3, presurge periods, or Days 8 and 10 post-treatment. At ovariectomy, 8 d post-estrus, more follicles > 10 mm diam. were observed in the ovaries after treatment with PMSG (8.5 +/- 5.66) than after treatment with FSH-P (0.7 +/- 0.42) (P<0.05). Maximum concentrations of PMSG were measured 24 h after administration. Following this peak, PMSG levels declined with two slopes, with half-lives of 36 h and 370 h.