The variability in the interval between estrus and ovulation in cattle and its determinants.

Fertility of Holstein cows has been decreasing for years and, to a lesser extent, the fertility of heifers too but more recently. A hypothesis to explain this phenomenon may be that the chronology of events leading to ovulation is different for those animals bred nowadays when compared to what was reported previously; this would result in an inappropriate time of insemination. Therefore, two experiments were designed to investigate the relationships among estrus behavior, follicular growth, hormonal events and time of ovulation in Holstein cows and heifers. In the first experiment, the onset of estrus, follicular growth, patterns of estradiol-17beta, progesterone and LH, and the time of ovulation were studied in 12 cyclic Holstein heifers that had their estrus synchronized using the Crestar method; this was done twice, 3 weeks apart. The intervals between estrus and ovulation, estrus and the LH peak, and between the LH peak and ovulation were, respectively, 38.5 h +/-3.0, 9.1 +/- 2.0 and 29.4 h +/-1.5 (mean+/- S.E.M). The variation in the interval between estrus and the LH peak explained 80.6% of the variation in the interval between estrus and ovulation. The intervals between estrus and the LH peak, and estrus and ovulation were correlated with estradiol-17beta peak value (r=-0.423, P <0.04 and r=-0.467, P<0.02, respectively). Positive correlation coefficients for the number of follicle larger than 5 mm, and negative correlation coefficients for the size of the preovulatory follicle with the intervals between estrus and LH peak, LH peak and ovulation, and estrus and ovulation suggest an ovarian control of these intervals. In respect to its role to explain the variation in the interval between estrus and ovulation, the variation in the interval between estrus and the LH peak was evaluated further in a second set of experiments utilizing 12 pubertal Holstein heifers and 35 Holstein cows. The duration of the interval between the beginning of estrus and the LH peak was longer in heifers than in cows (4.15 h versus -1.0 h; P <0.002); the variation for this interval was higher in cows than in heifers (S.E.M.= 1.2 h versus 0.8 h; P=0.01). According to the results of these studies it can be proposed that estradiol and other product(s) of ovarian origin regulate not only the duration of intervals between the onset of estrus and the LH surge but also between the LH surge and ovulation. From the results obtained in the first experiment, it may be postulated that differences observed between cows and heifers for the duration of the interval between onset of estrus and the LH surge as well as for the variation of this interval would be observed also for the interval between the onset of estrus and ovulation. Therefore, on a practical point of view, the long interval between the onset of estrus and ovulation and the high variation of this interval, especially in cows, may be a source of low fertility and should be considered when analysing reproductive disorders.

Relationships between calf birth weight, prepartum concentrations of plasma energy metabolites and resumption of ovulation postpartum in Limousine suckled beef cows.

The aim of this study was to determine the relationship between energy status before calving and calf birth weight and their potential effects on interval between calving and first ovulation. Sixty-nine Limousine, suckled beef cows were sampled weekly over a 3-yr period during the last 2 m.o. of pregnancy to determine the concentrations of nonesterified fatty acids (NEFA), beta-3-hydroxybutyrate (beta-OHB), glucose and glycerol. After parturition, progesterone concentrations were measured weekly to determine time of resumption of ovulation. Cows were allotted to 3 groups according to calf birth weight (Heavy: > 44 kg, n = 37; Medium: 39 to 43 kg, n = 56; and Light: < 38 kg, n = 45) and to postpartum ovarian resumption of cyclicity (Late: > 11 wk, n = 41; Mid: 7 to 10 wk, n = 57; and Early: < 6 wk, n = 40). Puerperium glycaemia of the dams was steady state (0.66 +/- 0.03 g/L) and was not related to calf birth weight. Plasma NEFA, beta-OHB and glycerol values were higher (P < 0.05) in Heavy than in Medium and Light group dams during the last 4 wk of pregnancy. Interval between calving and first ovulation was significantly longer for primiparous than for multiparous cows (respectively, 9.9 +/- 2.0 and 7.7 +/- 1.4 wk; P < 0.05). Calf birth weight was not related to time of first ovulation. Late primiparous cows had higher NEFA plasma concentrations than Mid and Early group primiparous cows during the last 4 wk of pregnancy, whereas NEFA plasma concentrations were not related to interval between calving and first ovulation in multiparous cows. Thus, lipomobilization increased with calf birth weight during the last 4 wk of pregnancy. High level of body reserves mobilization was associated with delayed first ovulation in primiparous but not in multiparous cows.

Serum cholesterol and triglycerides in postpartum beef cows and their relationship to the resumption of ovulation.

The variations in lipid metabolism according to the physiological stage and their relationship to the resumption of postpartum ovarian cyclicity were assessed in Limousine beef cows fed a grass diet over 3 yr. Weekly blood samples were collected from 59 cows beginning 10 wk before to 20 wk after calving to evaluate serum cholesterol and triglyceride concentrations and electrophoretic lipoprotein fractions. After parturition, progesterone concentrations were also measured at weekly intervals to determine time of resumption of ovulation. Cows were categorized by resumption of postpartum ovarian cyclicity into 3 groups: early (4 to 6 wk post partum, n = 36); mid (7 to 10 wk post partum, n = 46) and late (after 11 wk post partum, n = 38). Higher serum triglyceride values (P<0.05) were observed during the last 10 wk of pregnancy (0.36+/-0.15 g/L) than during the first 20 wk of suckling (0.29+/-0.09 g/L). Cholesterol values decreased significantly (P<0.05) at the end of pregnancy, were minimal (1.01+/-0.03 g/L) at parturition, and increased again up to 9 wk post calving. Increased cholesterolemia and low serum triglyceride values after calving could be linked to the increased bovine alpha-lipoprotein fraction and decreased beta fraction. Serum triglyceride concentrations were not related to the resumption of postpartum ovarian cyclicity. Higher serum cholesterol values were observed from 2 wk before to 4 wk after calving in cows with early rather than mid and late resumption of ovarian cyclicity. Therefore, modifications in lipid metabolism during the puerperium seem to be related to resumption of cyclicity during the early postpartum period.

Anti-Müllerian hormone (AMH) secretion in prepubertal and adult rams.

The aim of the present analysis was to determine whether anti-Müllerian hormone concentrations in prepubertal plasma or adult rete testis fluid are related to the number or function of Sertoli cells in rams or to the presence of the FecB Booroola gene. Twenty rams from two Booroola crosses, differing in their testicular masses were analysed; in each cross, half of the animals were heterozygous carriers of the FecB gene. The data from rams, during prepuberty and at adulthood during the non-sexual season, were analysed by two-way ANOVA and residual correlations. In 4-week-old intact male lambs, the mean anti-Müllerian hormone plasma concentration was 15 ng ml-1, irrespective of cross, genotype or eCG stimulation; it was significantly negatively correlated with FSH (r = -0.51; P = 0.02; n = 19). In adults, anti-Müllerian hormone was not detectable in plasma and was 0.5 ng ml-1 in rete testis fluid, irrespective of cross or genotype. The total number of Sertoli cells per testis was not related to anti-Müllerian hormone concentration in lamb prepubertal plasma or in adult rete testis fluid. The concentration of anti-Müllerian hormone in adult rete testis fluid was significantly and negatively correlated with the daily production of leptotene primary spermatocytes per testis (r = -0.56; P = 0.02; n = 16). The mean oestrogen concentration in the adult testicular vein was 2 pg ml-1 and was correlated negatively with the rete testis fluid concentration of anti-Müllerian hormone (r = -0.60; P = 0.02; n = 15) and correlated positively with the daily production of leptotene primary spermatocytes per testis (r = 0.53; P < 0.05; n = 19). In conclusion, anti-Müllerian hormone secretion was not correlated with the total numbers of Sertoli cells per testis and cannot be used as a predictor of the number of Sertoli cells. Anti-Müllerian hormone secretions were not affected by the presence of FecB gene. However, anti-Müllerian hormone secretion could be considered to be inversely related to the daily production of primary spermatocytes by the testis.

Estrus synchronization in dairy goats: use of fluorogestone acetate vaginal sponges or norgestomet ear implants.

The ultimate aim of any estrus synchronization method is to allow artificial insemination at a predetermined time after the end of treatment. This requires a very tight synchronization of estrus which is not observed in goats after administration of the usual fluorogestone acetate (FGA)/prostaglandin (PG) F2 alpha/equine chorionic gonadotrophin (eCG) treatment. The possibility to improve the synchronization of estrus and luteinizing hormone (LH) peak with different progestagens (FGA versus norgestomet) and routes of administration (vaginal sponge versus subcutaneous ear implant) was evaluated in two experiments where goats received one of three progestagen treatments: (1) a vaginal sponge impregnated with 45 mg of FGA, (2) a half-implant of norgestomet, or (3) a whole implant containing 3 mg of norgestomet. The progestagens were left in place for 11 days and intramuscular injections of 400 or 500 IU of eCG (according to milk yield) and 50 micrograms of an analogue of PGF2 alpha (cloprostenol) were given 48 h prior to progestagen removal. In Experiment 1, 117 cycling goats were checked for the time of onset of estrus, preovulatory LH peak and ovulation rate following estrus synchronization treatment. Goats treated with half-implants came into estrus earlier than those receiving vaginal sponges (27.8 +/- 5.0 h vs. 33.0 +/- 6.6 h, respectively; P < 0.05). No effect of progestagen priming was observed on the variability of the onset of estrus. However, the interval between the time of onset of estrus and LH peak was more variable (P < 0.05) in goats treated with half-implants. In Experiment 2, 170 non-cycling goats were monitored for the time of onset of estrus, percentage of females ovulating, fertility and prolificacy after estrus induction treatment and artificial insemination with frozen-thawed semen performed 24 h after the onset of estrus. No effect of progestagen treatment was observed either on the time or the variability of onset of estrus. The percentage of goats ovulating and overall fertility rate were higher (P < 0.05) in goats receiving vaginal sponges (98.2% and 75.0%, respectively) than those treated with half-implants (81.8% and 45.5%, respectively). However, no significant difference was observed, for the same parameters, in animals receiving implants (86.3% and 58.8%, respectively). In conclusion, estrus synchronization with a norgestomet implant or half-implant did not reduce the variability in the onset of estrus and LH peak. The fertility tended to be lower in goats treated with a whole implant and was significantly decreased in goats which received a half-implant.

Physiological limits to further improvement in the efficiency of oestrous synchronization in goats.

The variability between animals in the timing of oestrus after administration of a synchronization treatment seems to explain the low rate of fertility in goats inseminated at a predetermined time after progesterone withdrawal. Two experiments were performed during the breeding season to test whether the variation was due to the exogenous hormone regime or to the endogenous physiology of the animals. Twenty-one goats were given a synchronization treatment consisting of a vaginal sponge impregnated with 45 mg of fluorogestone acetate (FGA) for 11 days associated with intramuscular injection of 400 I.U. of equine chorionic gonadotrophin (eCG) and 50 microg of cloprostenol 48 h before sponge removal. Progesterone concentrations were measured during the subsequent cycle and the patterns were modelled to allow precise determination of the onset of luteolysis. Oestrus and the luteinizing hormone (LH) surge began 33.0+/-6.8 h and 76.0+/-33.0 h after sponge withdrawal, v. 43.4+/-5.7 h and 90.0+/-36.0 h after natural luteolysis. For both observations, the between-goat variability was larger during the natural than during the synchronized oestrus (P < 0.05). The duration of the oestrous cycle was independent of the number of corpora lutea (CL), whereas the duration of luteal phase was shorter in goats with 2-3 CL (16.4+/-0.9 day than in those with 1 CL: 17.7+/-1.3 day; P < 0.05). In the second experiment, 20 goats were ovariectomized and given a vaginal sponge as described above. Sixteen h after sponge removal, they were injected with 50 microg of oestradiol benzoate (ODB). This treatment was repeated with the second sponge being inserted 1-2 days after observation of oestrus. Oestrus and LH surge were observed: 32.8+/-6 8 h v. 27.8+/-7.8 h after the first ODB injection, and 36.6+/-7.3 h v. 34.3+/-4.8 h after the second ODB injection. No relationship was observed between data of the two experiments. In both cases, the variability in the occurrence of oestrus and LH surge was of the same order as observed in the first experiment. This study shows that the timing of oestrus and LH surge is less variable after progestagen treatment than during a natural oestrous cycle. Moreover, a significant proportion of variability is inherent in the delays following the oestradiol peak, suggesting that further improvement in the synchronizing capacity of treatment based on progestagen administration is unlikely.

Induction and synchronization of estrus in goats: the relative efficiency of one versus two fluorogestone acetate-impregnated vaginal sponges.

The purpose of the experiment was to test the hypothesis that a variable and/or insufficient level of progestagen at the end of a treatment to synchronize estrus in goats could explain variability in the onset of estrus. The experiment was performed during the anestrous season on 2 herds, one of Alpine (n = 49) the other of Saanen (n = 53) dairy goats. The animals were allocated to 1 of 3 treatments: Group 1 received a vaginal sponge impregnated with 45 mg of fluorogestone acetate (FGA) on Day 0; Group 2 received a sponge on Day 0 plus a second sponge on Day 7; Group 3 received a sponge on Day 0 plus a second sponge on Day 9. The sponges were withdrawn on Day 11. All goats received 400 or 500 IU eCG and 50 mug PGF(2alpha) analog 48 h prior to sponge removal. They were inseminated with frozen-thawed semen 24 h after the onset of estrus. Among treatment groups no difference (P > 0.05) was observed for the following parameters: percentage of goats in estrus, percentage of goats ovulating, mean time and variability of onset of estrus. The fertility of Alpine goats in Group 3 was significantly decreased (P < 0.05). No effect on prolificacy was noticed. These observations show that to increase progestagen level at the end of treatment did not improve estrus synchronization. They provide further evidence that treatments with too high progestagen amounts can decrease fertility.

A new method for controlling the precise time of occurrence of the preovulatory gonadotropin surge in superovulated goats.

In goats treated to induce superovulation, insemination at a predetermined time after the end of progestagen treatment leads to a low fertilization rate. To solve this problem we developed a new treatment based on the control of the occurrence of the endogenous LH peak with a GnRH antagonist (Antarelix). The first experiment was designed to determine the dose of LH required to mimic a spontaneous LH preovulatory discharge; the injection of 3 mg, i.v. of pLH induced a peak of the same amplitude and duration as the spontaneous peak. Subsequently, in the second experiment, we compared 2 doses of Antarelix (0.5 and 1 mg, sc) administered 12 h after sponge removal (9 goats/treatment group). The dose of 0.5 mg was selected for further experiments because it was effective in the inhibition of the endogenous LH peak and had no detrimental effect on the quality of embryos. In the final experiment, 48 goats received the new treatment and were inseminated (intrauterine) only once 16 h after LH injection; 41 were flushed and produced 5.3 +/- 4.5 (m +/- SD) transferable embryos. The developmental stage and the number of cells/embryo were within the range that has been reported for embryos produced with conventional treatments. In conclusion, with the described method, it is possible to inseminate goats at a predetermined time without decreasing the number of transferable embryos. This technique will encourage the development of embryo transfer within genetic programs, and it will be a valuable tool for the production of zygotes for gene transfer.

Structure of the bovine follicle-stimulating hormone receptor complementary DNA and expression in bovine tissues.

We report the complementary DNA structure obtained by reverse transcription and polymerase chain reaction amplification encoding the complete amino acid sequence for the bovine follicle-stimulating hormone receptor (bFSHr). The deduced amino acid sequence for the cDNA revealed a mature polypeptide consisting of 678 amino acids (theoretical weight of 76.4 kDa) and a 17 amino acid putative leading signal peptide. The receptor consists of a large NH2-terminal extracellular membrane domain of 349 aa with 3 potential N-linked glycosylation sites, a transmembrane domain (264 aa) consisting of 7 putative membrane spanning segments, and an intracytoplasmic COOH-terminal domain (65 aa). Four potential phosphorylation sites were found in the transmembrane domain and the COOH-terminal domain. The amino acid sequence is 97%, 89%, and 88% homologous to the ovine, human, and rat FSHr respectively, with complete conservation of the 22 cysteine residues in the whole protein and the 3 N-linked glycosylation sites on the extracellular membrane domain. Northern blot analysis of total mRNA in bovine tissues revealed a major mRNA transcript of 2.55 kb for the bFSHr in the ovary without corpus luteum, and in the testis. No expression was found in other tissues analyzed. Total RNA from bovine granulosa cells collected from pregnant mare serum gonadotropin (PMSG)-treated prepubertal heifers showed 2 major mRNA transcripts of 6.8 and 2.55 kb, and 3 minor transcripts of 3.8, 3.3, and 1.6 kb. Bovine granulosa cells cultured with porcine FSH (0, 2, 10 ng/ml) for 4 days showed a decrease in the steady state level of the FSHr mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

[Use of GnRH to stimulate the reproductive function in domestic ruminants]. / Utilisations de la GnRH dans la stimulation de la fonction de reproduction chez les ruminants domestiques.

Therapeutic effects of GnRH or its analogs have been tested in various physiological and pathological situations in domestic ruminants. Without any previous selection based on clinical observations, these treatments have a low efficiency to improve reproductive parameters. When used to induce ovulation in anoestrous females, GnRH or its analogs are less efficient than progestagen treatments associated with PMSG. On the contrary, GnRH can be used in females bearing follicular cysts or for the treatment of repeat breeders. Association of GnRH or its analogs to superovulation treatments is a new field of investigation. Additional experiments are necessary to show if it is possible to improve the response of superovulated females with such treatments.

Synchronization of estrus in goats: the relationship between time of occurrence of estrus and fertility following artificial insemination.

The fertility rate for goats following artificial insemination (AI) is usually analyzed according to herd or treatment groups. However, these general information are insufficient to allow identification of specific factors which affect this individual reproductive performance. In the present experiment 640 dairy goats were used to analyze to what extent the interval from sponge removal to estrus affects the results of AI, performed at a predetermined time following sponge removal. Estrus occurred in 98.1% of experimental animals between 24 and 72 hours after sponge removal. The fertility rate was lower for goats that came into estrus later than 30 hours after sponge removal (33.3%, n = 108 than for goats that exhibited estrus earlier (65.0%, n = 520; P<0.001). The occurrence of late estrus is not age dependent, but it increases with the number of treatments that an individual animal has previously received. These results show that the low fertility rate observed in some herds after synchronization of estrus and AI may be related to the high proportion of goats with a late occurrence of estrus, and this phenomenon increases in animals that are treated repeatedly.

The form and function of the Leydig cells in hypophysectomized rams treated with pituitary extract when spermatogenesis is disrupted by heating the testes.

The morphology and in-vivo function of the Leydig cells were studied in rams when spermatogenesis had been disrupted by a single exposure of the testes 20 days earlier to a temperature of about 42 degrees C for 45 min. To avoid complications due to changed negative feedback from the testes to the pituitary with consequent changes in the degree of gonadotrophic stimulation, ten of the animals (five heated and five unheated) were surgically hypophysectomized when the testes were heated and then treated twice daily with pituitary extract. Six intact rams (three heated and three unheated) were also studied. The heat-affected testes were about half the size of the unheated testes, and blood plasma flow was closely related to testis weight. There were no differences in the testosterone concentrations in spermatic venous blood, testicular lymph or rete testis fluid, or in oestradiol in spermatic venous plasma from heated or unheated testes. Consequently, testosterone secretion by the heat-affected testes was markedly reduced, and the concentrations in jugular blood were also lower in the heat-affected rams than in controls. The volume of the interstitial tissue was less in absolute terms in the heat-affected rams, but it made up a greater fraction of the testes. The absolute volume of the blood plus lymph vessels, and their fraction of the interstitial tissue were lower in the heat-affected testes, although there was no effect on their volume as a fraction of the whole testis. The heat-affected testes of the hormone-treated rams had fewer Leydig cells, but each cell was larger; no equivalent difference was found in the intact rams. However, the dose of pituitary extract chosen was somewhat excessive, as there were higher than normal concentrations of FSH, LH and testosterone in jugular blood plasma, of testosterone and oestradiol in testicular venous blood plasma and of testosterone in rete testis fluid in the hormone-treated hypophysectomized rams. The testes of the unheated hypophysectomized rams increased in size by about 20% during treatment with pituitary extract, although testicular blood plasma flow was lower per unit weight of testis. The absolute volume of each Leydig cell and the total volume in absolute terms and as a fraction of the interstitial tissue was greater in the hormone-treated than in the untreated rams, but not the volume as a fraction of the whole testis. The total number of Leydig cells was higher in the hormone-treated unheated rams than in all the other rams taken together.(ABSTRACT TRUNCATED AT 400 WORDS)

Are antibodies responsible for a decreased superovulatory response in goats which have been treated repeatedly with porcine follicle-stimulating hormone?

Repeated administration of xenogenic gonadotropins in human or animal species may be responsible for antibody production and refractoriness. An experiment was conducted in which goats were treated with porcine FSH (p-FSH) at 6-week intervals for a period of 7 months. A sensitive radioimmunoassay (RIA) was used to detect antibodies to p-FSH in plasma samples taken at short-term intervals during a 7-month period. Antibodies appeared after the first injection, and levels increased following booster injections. A high correlation rate existed between antibody level and superovulatory response. Refractoriness in goats was associated with a high level of antibodies.

Culture of bovine granulosa cells in a chemically defined serum-free medium: the effect of insulin and fibronectin on the response to FSH.

Granulosa cells from fully differentiated bovine follicles were cultured in serum-free medium for 4 days. At the end of culture, the number of viable cells was low (10-15% of cells plated on day one) and only progesterone secretion responded to FSH. Insulin increased the number of viable cells at the end of culture (ED50 # 70 ng/ml) and stimulated progesterone secretion (ED50 # 50 ng/ml); the secretion of oestradiol-17 beta over basal value was evident only for concentrations of 1000 and 10,000 ng/ml. FSH acted synergistically with insulin to modify steroid secretion. In the presence of 50 ng/ml of insulin, dose-response studies indicated that secretion of progesterone was maximal at 10 ng/ml of FSH and plateaud thereafter, while oestradiol output peaked at 2 ng/ml of FSH, decreasing at higher concentrations. When cells were seeded in wells precoated with fibronectin, a comparison with cells cultured on plastic showed an increase (30-40%) in the number of viable cells at the end of culture and in oestradiol secretion but a decrease in progesterone output. These results indicate that granulosa cells from large bovine follicles, cultured in a serum-free medium containing insulin, maintain their steroidogenic potency for at least 4 days. Moreover, they show that oestradiol and progesterone synthesis are differentially sensitive to FSH concentrations and that fibronectin increases oestradiol secretion in response to FSH.

Seasonal and hormonal control of pulsatile LH secretion in the dairy goat (Capra hircus).

In Exp. 1, the changes in pulsatile LH secretion at the onset of the breeding season were observed in 20 intact, mature Saanen does. Blood was sampled every 20 min for 6 h each week from the beginning of August until the onset of ovulatory activity, as evidenced by cycles in plasma progesterone. The first doe ovulated at the end of August and all were cycling by the end of September. As the first ovulation approached, LH pulse frequency increased by 67% and mean levels of LH increased by 47%. These changes were progressive rather than abrupt. In Exp. 2, seasonal changes in the inhibition of pulsatile LH secretion by ovarian steroids were studied in ovariectomized Saanen does. The animals were untreated (N = 4) or given subcutaneous oestradiol implants (N = 4) and blood was sampled every 10 min for 6 h, twice during the breeding season and twice during the anoestrous season. In each season, the second series of samples was taken after the animals had been treated with progesterone, administered by intravaginal implants. Season did not significantly affect LH secretion in goats not treated with oestradiol, but LH pulse frequency was 54% lower during the anoestrous season than during the breeding season in oestradiol-treated goats. Mean LH concentrations were affected in the same manner as pulse frequency, but pulse amplitude was increased by oestradiol treatment in both seasons. Progesterone had no detectable effect on LH secretion in either season. In Exp. 3, the response to repeated melatonin injections at a set time after dawn was investigated in 11 oestradiol-treated, ovariectomized goats.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of superovulation in cyclic heifers: The inhibitory effect of large doses of PMSG.

In two different experiments, superovulation was attempted with a PMSG-PG treatment; a bovine anti-PMSG serum was injected at estrus. After 2500, 5000 and 7500 IU of PMSG injected during the luteal phase, the mean ovulation rates were respectively 16.2 +/- 7.7, 3.2 +/- 2.1, and 1.4 +/- 0.6 in the first experiment (17 heifers) and 18.3 +/- 12.6, 8.5 +/- 8.2, and 2.2 +/- 2.3 in the second (19 heifers). The estradiol-17beta and progesterone patterns and the observations of the ovaries on the day of estrus (Day 0) by ultrasonic echography and on Day 8 by endoscopy show that the ovaries were highly stimulated and suggest that the inhibition observed with the largest doses reflects the absence of the preovulatory LH discharge or its effect.

Superovulation in the cow: comparison of oestradiol-17 beta and progesterone patterns in plasma and milk of cows induced to superovulate; relationships with ovarian responses.

Free oestradiol-17 beta, free + conjugated oestradiol-17 beta (total oestradiol-17 beta) and progesterone in milk, and free oestradiol-17 beta and progesterone in plasma were measured in 16 cyclic cows injected with FSH to induce superovulation during the treatment and periovulatory periods. The patterns of steroid secretion were the same in milk as in plasma but at different concentrations. Among oestrogens, the highest concentrations were measured for total oestradiol-17 beta in milk, followed by free oestradiol in plasma and free oestradiol in milk. Progesterone concentrations in milk were higher than in plasma. The peak concentrations of oestrogens were related to ovulation rate: Spearman Rank Correlation coefficient (r.s.) = 0.87 (P less than 0.001), 0.78 (P less than 0.001) and 0.69 (P less than 0.001) for total oestradiol, free oestradiol in milk and free oestradiol in plasma respectively. The increase in progesterone concentrations in milk between the beginning of treatment and prostaglandin injection was negatively correlated with the percentage of viable embryos among those recovered (r.s. = -0.68; P less than 0.001). This was not observed for progesterone in plasma. These results therefore show that the steroid pattern in milk gives a better indication as to the ovarian response to a superovulatory treatment than does the steroid pattern in plasma. In addition the fact that milk samples are easier to obtain and handle than blood plasma have led us to conclude that, to follow the effect of gonadotrophin stimulation, it would be more informative to assay oestradiol-17 beta and progesterone in milk rather than in plasma.

Changes in milk and plasma concentrations of progesterone in cows after treatment to induce superovulation and their relationships with number of ovulations and of embryos collected.

Progesterone concentrations in the milk of 86 Friesian cows induced to superovulate with an FSH-Cloprostenol treatment were studied daily from the day of estrus (D 0) to Day 7 (D 7). From D 2, a significant correlation between progesterone concentrations and ovulation rate was observed. Such a relationship was also observed beginning to D 3 between progesterone concentrations and the number of embryos recovered. No relationship was found between progesterone content and the number of viable embryos. For 30 of these cows, progesterone concentrations in blood plasma were also studied. The hormonal patterns in plasma and milk were similar but quantitative relationships were demonstrated earlier for progesterone in plasma than for progesterone in milk. It is concluded that relationships between milk progesterone concentrations and ovarian responses to a superovulatory treatment exist and could be of interest in embryo transfer programs in when predicting the number of embryos to be recovered.

A double antibody radioimmunoassay for free and conjugated estradiol-17 beta in cow's milk.

A radioimmunoassay for free estradiol-17 beta, conjugated estradiol-17 beta or total (free + conjugated) estradiol-17 beta in defatted milk of cows is described. Conjugated estradiol-17 beta was hydrolyzed by enzymes of Helix pomatia juice. Estrogens were extracted with dichloromethane; no other purification step was required before radioimmunoassay because of the high specificity of the antiserum. Immunoprecipitation was used to separate bound and free estradiol-17 beta. Concentrations measured were corrected for procedural losses on a per sample basis. The assays were shown to be accurate and specific. The sensitivity was 1.3pg/ml for the assay of free estradiol-17 beta (5ml of milk extracted) and 2.9pg/ml for conjugated or total estradiol-17 beta (2 ml of milk hydrolyzed and extracted). Estrogens were measured in the milk of cyclic cows and in cows stimulated with pregnant mare serum gonadotropin (PMSG). A preovulatory increase was clearly observed. Wether or not the ovary was stimulated by PMSG, concentrations of estrogens were higher and the relative increase during the preovulatory peak was greater for conjugated estradiol-17 beta than for the free form. The assay of conjugated or total estradiol-17 beta in defatted milk should be a practical method for assessing preovulatory growth of follicles in cows.

Effect of injection time of anti-PMSG antiserum on ovulation rate and quality of embryos in superovulated cows.

Eighteen cows were superovulated by injecting 3000 IU of PMSG during the luteal phase, followed 48h later with an injection of Estrumate. They were then placed in a control group or were given anti-PMSG antiserum at either 12h or 24h after the onset of oestrus. Sixteen of these animals were used for the same experiment five months later. The results were pooled because they were not significantly different between the two treatment periods. The timing of the injection of anti-PMSG antiserum, either 12h (11 cows) or 24h (12 cows) after the onset of oestrus, did not significantly affect the ovulation rate, the number of embryos collected or the number of good embryos. The antiserum significantly increased the number of good embryos but did not affect the ovulation rate or embryo recovery. It is concluded that even with a moderate dose of PMSG, the use of anti-PMSG at 12h or 24h after the beginning of oestrus improves the quality of embryos. The mean number of embryos to be transferred (5.5) is in the range of those obtained after the FSH treatments, but the procedure required only three injections compared with nine for the FSH treatment.