Effect of norgestomet treatment after insemination on the calving rate of postpartum suckled beef cows

Two experiments were conducted on postpartum suckled beef cows synchronized with Syncro-Mate B and artificially inseminated approximately 48 h after implant removal. In Exp. 1, cows (> or = 42 d postpartum at the timed AI) were randomly assigned to treated (n = 101) and control (n = 85) groups on d 12 after the timed AI. Treated cows received norgestomet/silicone implants that were left in situ for 9 d. Norgestomet treatment had no effect (P > .25) on the calving rates from the initial timed AI or from the return estrus. Nonpregnant norgestomet-treated cows returned to estrus in a more (P < .05) synchronized manner than the nonpregnant control cows. In Exp. 2, early postpartum cows (< 42 d postpartum at the first AI; n = 30) were included and all 118 cows (88 cows were > or = 42 d postpartum) received norgestomet/silicone implants as in Exp. 1. Of the 30 early postpartum cows, eight (19 to 41 d postpartum at the time of the first AI; mean = 29.3 d) calved to the first AI and nine calved to the second synchronized estrus. The calving dates at the next calving season for these 17 cows (57% of the cows in this group) was advanced an average of 46 d (319-d calving interval). The calving rates for the two timed insemination periods were similar (P > .25) for early and later (> or = 42 d postpartum) postpartum cows. Treatment with norgestomet implants on d 12 through 21 had no detrimental effects on established or subsequent pregnancy, synchronized the return estrus of nonpregnant cows, and was efficacious in establishing pregnancy early postpartum.     

Effect of nutritional management, trace mineral supplementation, and norgestomet implant on attainment of puberty in beef heifers

We conducted a study to evaluate the influences of nutritional management, trace mineral supplementation, and exogenous progesterone on attainment of puberty in beef heifers. Heifers (n = 180) were assigned at weaning to blocks and treatments. Treatments included two dietary regimens (corn silage vs pasture + oatlage), trace mineral supplementation, and puberty induction strategy (with or without progestin implant). Heifers that received pasture + oatlage were managed on grass-legume pastures from October 14 until December 14 and were then placed in pens and fed an oatlage-based diet through May 1994. Heifers fed the corn silage-based diet were housed in pens throughout the study. Norgestomet was implanted in half of the heifers on April 11 for 10 d. Progestin implant increased (P < .05) the number of heifers that had attained puberty by the end of the study, compared with nonimplanted heifers (89% vs 71%). Trace mineral supplementation did not affect percentage of heifers that reached puberty before the implant period. Plasma copper levels were below recommended levels in heifers fed oatlage-based diets without trace minerals. We conclude that heifers can be placed on regrowth in irrigated pastures during the fall and still make acceptable gains for attainment of puberty the following spring and that progestin treatment can aid in inducing heifers to reach puberty.     

Gonadotropin-releasing hormone axons target the median eminence: in vitro evidence for diffusible chemoattractive signals from the mediobasal hypothalamus

The projection of GnRH neurons to the median eminence of the medial basal hypothalamus (MBH) is established early in development and is also seen when preoptic area-derived GnRH cell-containing grafts are placed in the third ventricle of hypogonadal mice. To further study the factors directing GnRH axonal targeting, we cultivated embryonic or postnatal day 1 preoptic area with a coexplant on collagen- and laminin-coated membranes in insert chambers. After 7 days of culture, GnRH-immunoreactive fibers extended significantly farther and in greater number onto the sector of membrane facing a MBH coexplant than in the opposite sector, but not toward coexplants of control tissue. Moreover, such effects were specific, as outgrowth of a general axonal population, immunoreactive for growth-associated protein 43 was not influenced by the presence of the MBH. Preferential GnRH outgrowth toward the MBH was established early and was maintained during 10 days of culture. The importance of substrate-derived guidance was also assessed with confocal microscopy. GnRH axons consistently traveled in the company of growth-associated protein 43-labeled axons, but only erratic associations were seen between GnRH and glial processes extending on the membrane. We suggest that although employing an axonal substrate, GnRH axons follow a diffusible chemoattractive signal(s) secreted by the MBH.     

Bordetella pertussis filamentous hemagglutinin enhances the immunogenicity of liposome-delivered antigen administered intranasally

In an attempt to increase the immunogenicity of mucosally delivered antigens, we incorporated the Bordetella pertussis filamentous hemagglutinin (FHA) adhesin into liposomes containing the glutathione S-transferase of Schistosoma mansoni (Sm28GST) as a model antigen. Outbred mice immunized twice intranasally with liposomes containing a constant suboptimal dose of Sm28GST and increasing doses of FHA produced anti-Sm28GST antibodies in a FHA dose-dependent manner. The addition of 3 microg of FHA to the liposomes induced more than 10-fold-higher anti-Sm28GST antibody titers, compared to those induced by liposomes without FHA. The presence of FHA did not alter the nature of the humoral immune response, and the sera contained anti-Sm28GST immunoglobulin G1 (IgG1), IgG2a, and IgG2b. However, anti-Sm28GST IgA was only detected when at least 3 microg of FHA was added to the preparation. These results show a promising potential for FHA to enhance the immunogenicity of mucosally administered antigens incorporated into liposomes.     

Gonadotropin-releasing hormone neuronal system of the freshwater snails Helisoma trivolvis and Lymnaea stagnalis: possible involvement in reproduction

Peptides of the gonadotropin-releasing hormone (GnRH) family are present in neural and nonneural tissues throughout the chordate phylum. Although GnRH peptides have been implicated in nonreproductive functions, their primary function is to control reproduction by regulating sexual behaviors and inducing gonadotropin hormone release from the pituitary. Evidence suggesting the presence of a similar peptide in the central nervous system (CNS) of the gastropod mollusc Helisoma trivolvis has recently been provided. In the present study, we examined the tissue distribution of the peptide and found that it is likely restricted to the nervous system. The neuronal system containing the endogenous GnRH-like peptide is described further and is shown, in part, to innervate the male reproductive tract. Immunostaining in the closely related snail, Lymnaea stagnalis, showed a conservation in the locations of some immunoreactive neurons. Notably, staining occurred in and adjacent to the lateral lobes of both snails. Because these lobes contain neurons involved in the stimulation of egg laying and GnRH staining occurred in additional areas in the Helisoma CNS that are involved in reproduction, we suggest that the endogenous GnRH-like peptide plays a role in regulating reproduction in freshwater snails.     

Evaluation of different protocols for prostaglandin synchronization to improve reproductive performance in dairy herds with low estrus detection efficiency

The objective of this study was to evaluate different PGF2 alpha protocols against control protocols for herds with estrus detection efficiencies of 35, 55, and 75% using modeling and simulation: 1) PGF2 alpha treatments based on the presence of a corpus luteum diagnosed by rectal palpation, 2) PGF2 alpha treatments based on the presence of a corpus luteum diagnosed by an on-farm milk progesterone enzyme immunoassay, and 3) PGF2 alpha treatments based on a 14-d fixed treatment schedule without prior screening for ovarian status. After the start of each protocol, estrus detection efficiency was 75% for 7 d after treatment and 35 or 0% for the following week. For the third protocol, an additional modification at estrus detection efficiencies of 85 and 55%, respectively, in the 1st and 2nd wk after treatment was evaluated to establish a protocol for best case assumptions. All protocols improved reproductive performance relative to that of controls with estrus detection efficiencies of 35 and 55%. The mean number of days open was reduced from 124.3 d in the control herd to 95.9, 95.0, and 92.7 for the protocols based on rectal palpation, milk progesterone test, and the fixed treatment schedule, respectively. The protocols based on a fixed treatment schedule were superior to protocols based on rectal palpation and on-farm milk progesterone tests and resulted in better reproductive performance and a higher increase in net return per cow per year. Relative to a control herd with an estrus detection efficiency of 55%, it was cost effective to spend up to $10 per dose of PGF2 alpha, $9 per milk progesterone test, and $6 per rectal palpation.     

Oral administration of arginine enhances the growth hormone response to growth hormone releasing hormone in short children

We have evaluated the effect of oral administration of arginine chlorhydrate on the growth hormone response to growth hormone releasing hormone in a group of nine short prepubertal children (six boys and four girls). Arginine chlorhydrate 10 g, administered orally 60 min before an i.v. bolus injection of growth hormone releasing hormone 1-29, 1 microgram/kg, significantly enhanced the growth hormone response to the neuropeptide, confirming the results of previous studies which used the i.v. route. Furthermore, our data strengthen the view that the effects of arginine chlorhydrate on growth hormone secretion are mediated by inhibition of endogenous somatostatin release.     

Gonadotropin-releasing hormone (GnRH) in ancient teleosts, the bonytongue fishes: putative origin of salmon GnRH

PURPOSE: To measure the compressive forces of the haptics of 28 intraocular lens (IOL) models for different modes of compression and compare the results of two types of measurements. SETTING: Department of Ophthalmology, Central Hospital of Central Finland, Jyv?skyl?, Finland. METHODS: The haptics of 28 types of IOLs were compressed to a diameter of 9.0 mm between curved anvils. The compression forces in the plane of compression (i.e., in the plane of the optics) were measured at 0.5 mm intervals. During compression, the optics and the haptics were free to rotate with respect to the anvils. The results were compared with those of earlier measurements in which the optics were held fixed during compression. Perpendicular forces were measured at 0.4 mm intervals. RESULTS: The measured forces in the plane of the optics varied between 114 and 659 mg at a diameter of 10.0 mm and 192 and 1047 mg at a diameter of 9.0 mm. When compressed to 10.0 mm in diameter, the forces were 1 to 75% lower than when lens rotation was not possible. The forces perpendicular to the optic varied between 0 and 96 mg at a 10.0 mm diameter and correlated with the forces in the plane of the optic. CONCLUSION: The compression forces of the lens haptics were generally lower when the lenses were allowed to rotate during compression. The orders of stiffness of the haptics in these two measurements were similar. The perpendicular forces were generally small and correlated significantly with the forces measured in the plane of the optic.     

Effect of prostaglandin F2 alpha treatment before norgestomet and estradiol valerate treatment on regression, formation, and function of corpora lutea in beef heifers

Despite the presence of several human disease genes on chromosome 11q13, few of them have been molecularly cloned. Here, we report the construction of a contig map encompassing 11q13.1-q13.3 using bacteriophage P1 (P1), bacterial artificial chromosome (BAC), and P1-derived artificial chromosome (PAC). The contig map comprises 32 P1 clones, 27 BAC clones, 6 PAC clones, and 1 YAC clone and spans a 3-Mb region from D11S480 to D11S913. The map encompasses all the candidate loci of Bardet-Biedle syndrome type I (BBS1) and spinocerebellar ataxia type 5 (SCA5), one-third of the distal region for hereditary paraganglioma 2 (PGL2), and one-third of the central region for insulin-dependent diabetes mellitus 4 (IDDM4). In the process of map construction, 61 new sequence-tagged site (STS) markers were developed from the Not I linking clones and the termini of clone inserts. We have also mapped 30 ESTs on this map. This contig map will facilitate the isolation of polymorphic markers for a more refined analysis of the disease gene region and identification of candidate genes by direct cDNA selection, as well as prediction of gene function from sequence information of these bacterial clones.     

Differences in natural steroid hormone patterns of beef from bulls and steers

This investigation gives an overview of the concentrations of naturally occurring androgens, progestogens, corticosteroids, and their precursors and metabolites in meat from bulls and steers. A recently developed gas chromatography-mass spectrometry IGC-MS) method with improved sensitivity for steroid analysis was used. Eighty-two beef samples were analyzed using the GC-MS method. Beef from bulls contained higher concentrations of testosterone, which is an anabolic androgen, and its metabolite epitestosterone (P < .01) and the androgen precursor dehydroepiandrosterone (P < .05) than beef from steers. Beef from steers contained higher (P < .05) concentrations of the basic hormone precursor pregnenolone and cortisol, which is a catabolic corticosteroid, than beef from bulls. A classification of an unknown beef sample to one of the categories (bull or steer) was possible in most cases (>90%) using a masculinity index (MI) that was calculated using the concentrations of testosterone, epitestosterone, and pregnenolone. Because the hormonal status of beef cattle is related to meat quality characteristics, such as tenderness or fat and protein distribution, the MI may contribute to meat quality assessment and meat quality control.     

Plasma concentrations of prolactin, growth hormone, and luteinizing hormone in steers administered ergotamine or ergonovine

This research investigated whether ergot alkaloids associated with endophyte-infected tall fescue could alter plasma concentrations of pituitary hormones that regulate biological processes related to cattle performance. Seven Angus yearling steers received single i.v. injections of ergotamine tartrate, ergonovine maleate, or saline vehicle in a simple cross-over design. Each steer was given a different compound each week. Blood samples were collected at 15-min intervals for 45 min before and 240 min after treatments to assess plasma concentrations of prolactin, growth hormone, and LH. Respiratory rates were measured hourly to ascertain a systemic effect. Ambient temperature averaged 34 degrees C during data collection. Treatment x time was a significant source of variation for respiration rate and plasma concentrations of each hormone evaluated. Respiration rates were higher for ergonovine than for saline (P < .02) and ergotamine (P < .07) 30 min after treatment, but they were higher (P < .05) for ergotamine than for ergonovine and saline by 210 min after treatment. Both alkaloids transiently elevated (P < .01) plasma growth hormone concentrations compared with before alkaloid treatment and after saline treatment. Ergotamine reduced (P < .01) plasma concentrations of prolactin and LH throughout the 120-min period after treatment compared with concentrations before ergotamine treatment and after saline treatment. Ergonovine lowered (P < .01) prolactin concentrations for a shorter time than ergotamine and did not affect mean LH concentrations. Results indicated that ergot alkaloids implicated as contributing agents to fescue toxicosis can alter plasma concentrations of pituitary hormones important to cattle production.     

Gonadotropin-releasing hormone immunoreactivity in the nasal epithelia of adults with Kallmann's syndrome and isolated hypogonadotropic hypogonadism and in the early midtrimester human fetus

GnRH-secreting neurons are known to originate in the epithelium of the medial olfactory placode, whence they migrate along the axons of the terminal nerve via the forebrain and into the hypothalamus. Synaptic contact between the developing olfactory bulbs and fascicles of the vomeronasal, terminal, and olfactory nerves does not occur in Kallmann's syndrome. Consequently, there is migration arrest of GnRH cells and partial or complete failure of formation of the olfactory bulbs, resulting in severe olfactory deficit and hypogonadotropic hypogonadism. In the present study, using an immunofluorescent, double immunostaining technique and confocal laser scanning microscopy, we observed GnRH-immunoreactive neurons in the hypothalamus of a 14-week-old human fetus. However, migration of GnRH neurons was not complete, and indeed, such cells were seen to be migrating along terminal nerve fascicles beneath the cribriform plate in a 16-week-old fetus. The same immunofluorescent technique demonstrated the presence of GnRH cells in biopsies of nasal mucosa obtained from three adults with Kallmann's syndrome, one normosmic subject with hypogonadotropic hypogonadism, and a eugonadal male cadaver. These findings are consistent with two different interpretations: the nasal GnRH neurons may be vestigial, representing cells that failed to migrate during embryogenesis; alternatively, they may have been generated de novo later in life, a possibility consistent with the recognized plasticity of human postnatal olfactory neuroepithelium. They also reveal that subjects with the normosmic (i.e. non-Kallmann's) form of GnRH deficiency are able to synthesize immunologically recognizable GnRH, implying that failure of GnRH synthesis is not responsible for this type of hypogonadotropic hypogonadism.     

Development of a progestin-based estrus synchronization program: I. Reproductive response of cows fed melengestrol acetate for 20 days with an injection of progesterone

We designed two experiments to determine the efficacy of an estrus control system in cows that combined long-term progestin exposure (20 d) with an acute increase in progesterone concentration. In Exp. 1, cows (n = 30) were fed either melengestrol acetate (MGA; .5 mg x cow(-1) x d(-1)) or ground ear corn (MGA carrier) for 20 d. On d -15 (last day of MGA feeding = d 0), cows were administered 25 mg of PGF2alpha to regress the corpus luteum (CL) and establish an environment conducive to the development of persistent follicles. To synchronously regress persistent follicles, cows fed MGA (n = 15) were injected with 200 mg of progesterone on d -2 (MGA-P), and the cows fed the MGA carrier were not treated (CONT; n = 15). Cows in the CONT group were artificially inseminated 12 h after detection of spontaneous estrus from d -20 to d 8. Estrus was observed, and all cows in the MGA-P group were artificially inseminated during the period of estrus synchronization (SYNC; d 1 to 8). No difference in conception rate was observed between treatments. In Exp. 2, postpartum cows (n = 113) received either the MGA-P (n = 56) or CONT (n = 57) treatment. More (P < .05) cows were observed in estrus during SYNC in the MGA-P (50%) than in the CONT (28%) group. Of the cows in the MGA-P group that were not observed in estrus during SYNC, 50% were in estrus for the first time 23 to 29 d after MGA withdrawal (SYNC2), suggesting that these cows ovulated without observable estrus during SYNC. Estrus was observed for the first time during SYNC2 in more (P < .05) cows in the MGA-P (25%) than in the CONT (7%) group. Conception rate at the synchronized estrus, pregnancy rate, and interval to first service and pregnancy were similar between treatments. We conclude that administration of MGA-P results in the synchronization and(or) induction of a fertile estrus in cows.     

Gonadotropin-releasing hormone receptors with intracellular carboxyl-terminal tails undergo acute desensitization of total inositol phosphate production and exhibit accelerated internalization kinetics

The mammalian gonadotropin-releasing hormone receptor (GnRH-R) is the only G-protein-coupled receptor (GPCR) in which the intracellular C-terminal tail is completely absent. In contrast to other GPCRs, the GnRH-R does not show rapid desensitization of total inositol (IP) production, and the rates of internalization are exceptionally slow. We investigated whether the incorporation of a cytoplasmic tail into the C terminus of the GnRH-R affects desensitization events and receptor internalization rates. A GnRH-R/TRH-R chimera was created where the intracellular tail of the rat thyrotropin-releasing hormone receptor (TRH-R) was engineered into the C terminus of the rat GnRH-R. Three different rat GnRH-R cDNA stop codon mutations (one for each reading frame) were also made. The GnRH-stimulated IP production of the wild-type rat GnRH-R expressed in either COS-7 or HEK 293 cells did not desensitize even after prolonged stimulation with GnRH. In contrast, the catfish GnRH-R (which does possess an intracellular tail) and the TRH-R rapidly (<10 min) desensitized following agonist stimulation. The GnRH-R/TRH-R chimera also desensitized following treatment with GnRH, resembling the pattern shown by the TRH-R and the catfish GnRH-R. Two of the stop codon mutants did not show desensitization of IP production, and the third mutant with the longest tail was not functional. Internalization experiments showed that the rat GnRH-R had the slowest endocytosis and recycling rates compared with the TRH-R, the catfish GnRH-R, and the chimeric GnRH/TRH-R. This study demonstrates that the addition of a functional intracellular C-terminal tail to the GnRH-R produces rapid desensitization of IP production and significantly increases internalization rates.     

An android body fat distribution in females impairs the pregnancy rate of in-vitro fertilization-embryo transfer

To assess if the waist:hip ratio (WHR) is associated with the pregnancy rate (PR) in in-vitro fertilization (IVF) and embryo transfer, waist and hip girths, in addition to height, weight, body mass index (BMI), indications for IVF, PR and other related variables, were measured in 220 women undergoing IVF-embryo transfer. Three variables were significantly negatively associated with PR; high age, smoking and WHR >0.80. Women with WHR between 0.70-0.79 had a PR of 29.9% as compared to 15.9% in women with WHR >0.80 [odds ratio 0.42, 95% confidence interval (CI) 0.2-0.9, P = 0.03]. There were no correlations between BMI and PR, nor were there any significant differences for the indications for IVF-embryo transfer, number of oocytes or oocyte fertilization rate, cleavage rate and number of embryos transferred. The association between a low PR and WHR >0.80 remained unchanged after adjustment for age, BMI, smoking, indication for IVF, parity and number of embryos transferred. In IVF-embryo transfer, fertilization is a laboratory and clinically controlled process, until the embryo is transferred to the uterus. Possible reasons for our finding of a decreased PR in women with an android body fat distribution include a different endocrinological and biochemical milieu for the oocyte in the growing follicle, oocytes of poor quality, or endometrial changes due to hormonal dysfunction.     

Effects of growth rate on carcass composition and lipid partitioning at puberty and growth hormone, insulin-like growth factor I, insulin, and metabolites before puberty in beef heifers

The effect of three rates of gain on carcass composition, lipid partitioning, age and BW at puberty, and concentrations of growth hormone (GH), IGF-I, insulin, glucose, and NEFA in plasma were evaluated in 38 Angus x Hereford heifers. Heifers were allotted by BW and age to three treatments with a replication in each of 2 yr: full-fed (n = 13; FF) to gain 1.36 kg/d; limit-fed (n = 12; LF) to gain .68 kg/d; maintenance-full-fed (n = 13; MFF) to gain .23 kg/d for 16 wk, then full-fed to gain 1.36 kg/d. Heifers were slaughtered within 10 d after the onset of puberty. At slaughter, kidney, pelvic, and heart fat (KPH) and udder (UDDER) were separated from carcass, as was fat surrounding viscera (OM). After 48 h at 4 degrees C a carcass side was dissected into subcutaneous fat (SC), intermuscular fat (SEAM), soft tissue (SFT = inseparable lean and fat), LEAN, and BONE. In yr 1, LF heifers (431 d) were older (P < .05) than MFF heifers (371 d) at puberty, but age of FF heifers (389 d) did not differ (P > .10) from that of LF and MFF heifers. In yr 2, FF heifers (351 d) were younger (P < .05) than LF and MFF heifers (398 and 434 d, respectively). The FF heifers had greater (P < .05) BW and a greater (P < .01) percentage of lipid in the carcass at puberty than LF and MFF heifers. During the first 16 wk of treatment, concentrations of NEFA were greater in heifers with slower daily gains (MFF > LF > FF; P < .01). Concentrations of NEFA were lesser and concentrations of IGF-I and insulin were greater in plasma of FF than in that of MFF heifers during the 10 wk before puberty. Treatment significantly altered age, BW, carcass composition, and lipid partitioning at puberty in beef heifers. We conclude that the percentage of body fat is not the sole regulator of puberty, and age may be an important modulator in determining the onset of puberty in beef heifers.     

Development of a progestin-based estrus synchronization program: II. Reproductive response of cows fed melengestrol acetate for 14 days with injections of progesterone and prostaglandin F2alpha

We tested the efficacy of an estrus control system designed to provide optimal control of follicular development. In Exp. 1, postpartum cows (n = 133) and yearling heifers (n = 57) were fed either .5 mg x female(-1) x d(-1) of melengestrol acetate (MGA) or the carrier for MGA from d -13 to d 0 (d 0 = last day of MGA feeding). All females received 25 mg of PGF2alpha (i.m.) on d -13 and 0. On d -6, cows and heifers fed MGA were administered an i.m. injection of progesterone (200 mg; MGA/P4), and those fed the corn carrier (2XPGF2alpha) received no progesterone. Beginning on d 1, females were bred by AI from d 1 to at least d 5. During the estrus synchronization period (d 1 to d 5), more (P < .05) postpartum cows were observed in estrus (70.1 vs 42.4%), the timing of estrus was more (P < .05) precise, conception rate was similar, and pregnancy rate was higher (P < .05) in the MGA/P4 than in the 2XPGF2alpha treatment. More (P < .05) cows that were anestrous at the beginning of the breeding season were in estrus during the synchronization period in the MGA/P4 (55.8%) than in the 2XPGF2alpha (28.6%) treatment. In heifers, estrus was synchronized in over 90% of females, and neither conception nor pregnancy rate during the synchronization period differed between treatments. In Exp. 2, postpartum cows (n = 122) and heifers (n = 84) received treatments (MGA/P4 or 2XPGF2alpha) as described for Exp. 1 with one exception. In the MGA/ P4 treatment, progesterone was administered on d -7 rather than d -6. Females were bred by AI from d 1 to 5. The estrus response and conception rate during the synchronization period did not differ between treatments for either cows or heifers. We conclude that the progestin-based estrous synchronization system used in this study effectively synchronized an estrus of normal fertility in cyclic cows and induced a majority of anestrous cows to reinitiate estrous cycles.     

Effect of time of artificial insemination on pregnancy rates, calving rates, pregnancy loss, and gender ratio after synchronization of ovulation in lactating dairy cows

In order to assess the optimal time of artificial insemination (AI) in relation to ovulation, lactating dairy cows (n = 732) from herds with rolling herd averages of 9980 to 11,800 kg from three milkings per day were randomly assigned to five groups by stage of lactation and parity. Ovulation was synchronized by administration of GnRH followed 7 d later with PGF2 alpha followed 2 d later with a second treatment with GnRH. Cows were inseminated at 0, 8, 16, 24, or 32 h after the second injection of GnRH (ovulation occurs between 24 and 32 h after GnRH). Pregnancy diagnoses were performed by ultrasound at 25 to 35 d post-AI. Pregnancy rates per AI were similar for the groups inseminated at 0, 8, 16, and 24 h and lower for the group inseminated at 32 h. A significant quadratic effect of treatment suggests that the middle time periods (8, 16, and 24 h) may produce the greatest pregnancy rate per AI. However, the group inseminated at 0 h had lowest pregnancy loss, and the group inseminated at 32 h tended to have the greatest pregnancy loss compared with that of the other groups. The calving rate was similar between the groups inseminated at 0, 8, 16, and 24 h and lower in the group inseminated at 32 h. The time of AI also appeared to affect gender of calf: cows bred at 0 and 32 h having a higher percentage of female offspring. In conclusion, there appears to be substantial flexibility in the time of AI after the second injection of GnRH, and lower reproductive rates were observed only when AI was after the time of ovulation.