Factors controlling epidermal growth factor (EGF) gene expression in the endometrium of the mare

Previous studies showed a dramatic increase in EGF gene expression in the endometrial glands of pregnant mares around day 40 after ovulation. To investigate how the steroid hormones of pregnancy might regulate this expression, in situ hybridization was used to monitor the levels of EGF mRNA in endometrial biopsies obtained from seasonally anoestrous or ovariectomised mares given exogenous progesterone and oestrogen, alone or in combination, for up to 46 days. Biopsies were also taken from mares during the non‐pregnant cycle, during normal pregnancies and pregnancies compromised by endometrial pathology (endometrosis) or because of incompatible extraspecific embryo transfers (donkey‐in‐horse pregnancies). Only a few samples showed weak EGF expression during the late luteal phase of the oestrous cycle. During normal pregnancy, the previously observed dramatic increase of expression after day 40 of gestation was confirmed. Although aged mares suffering from endometrosis and mares carrying an extraspecific donkey conceptus showed the same increase of EGF mRNA in normal glands, this was virtually absent from gland cross‐sections compromised due to inflammatory or fibrotic changes. Administration of various doses and combinations of progesterone and oestrogen for <35 days yielded negative or only weakly positive hybridization results, whereas progesterone alone for ≥40 days upregulated EGF expression strongly irrespective of additional treatment with oestrogen. This is the first experimental evidence that EGF expression in the endometrium can be induced by progesterone alone. The requirement for prolonged progesterone priming is of considerable interest in the context of the unusually late stage of gestation at which placental attachment commences in equids. Mol. Reprod. Dev. 53:255–265, 1999. © 1999 Wiley‐Liss, Inc.     

Cell proliferation patterns in the equine endometrium throughout the non-pregnant reproductive cycle.

Immunohistochemical detection of the proliferation marker Ki-67 antigen was used to monitor mitotic activity in the endometrium of mares. The monoclonal antibody MIB1 was validated for use on equine tissues by demonstrating its reaction with activated peripheral blood lymphocytes, and endometrial biopsies were recovered from 26 non-pregnant mares at selected stages during the reproductive cycle. The proportion of positively stained nuclei was counted in five random areas on each histological section to determine the percentage and type of proliferating cells. Multiplication rates in the types of cell found in the superficial strata, comprising the luminal epithelium, the epithelium of the gland necks and the stromal cells of the stratum compactum, were greatest during oestrus, presumably under the influence of oestrogens secreted by the growing ovarian follicles. In contrast, the mitotic activity in the cells of the deeper secretory portions of the endometrial glands was restricted to a brief phase between day 3 and day 7 of dioestrus, most likely as a delayed response to the decreasing oestrogen concentrations after ovulation. Some of the degenerate glands in subfertile mares did not follow this pattern of increased epithelial proliferation at that stage. After day 7 of dioestrus, the proliferation rates of cells in the endometrium decreased to basal values and remained low for as long as progesterone concentrations remained evaluated, even during prolonged dioestrus. The technique enabled characterization of normal cell proliferation patterns in the endometrium of mares and it will be a useful tool in the future for monitoring the endometrial responses of reproductively healthy and subfertile mares.     

Effect of ovariectomy on pregnancy in mares.

Pony mares were bilaterally ovariectomized at different stages of pregnancy between Days 25 and 210. Abortion or fetal resorption occurred within 2 to 6 days after operations in all 14 mares ovariectomized between Days 25 and 45 and after an interval of 10 to 15 days in 9 of 20 other ovariectomized between 50 and 70 days. All 12 mares ovariectomized on either 140 or 210 days carried their foals to normal term. The termination of early pregnancy was preceded by a loss of uterine tone and of a palpable uterine bulge. The mean length of gestation in all mares in which pregnancy was not interrupted by ovariectomy was not significantly different from that in a group of contemporary control mares. Plasma progestagen concentrations dropped to less than 2 ng/ml after ovariectomy, whether or not pregnancy was maintained. Mares ovariectomized on Day 25 and injected with 100 mg progesterone daily for 10 or 20 days remained pregnant during treatment but showed a loss of uterine tone and the fetal bulge disappeared within 4 to 6 days after the end of treatment. Non-pregnant ovariectomized or intact seasonally anoestrous mares injected i.m. with 50 or 100 mg progesterone daily for 8 weeks showed changes in uterine tone, length and thickness similar to those occurring in mares during early pregnancy.     

Changes in equine endometrial oestrogen receptor alpha and progesterone receptor mRNAs during the oestrous cycle, early pregnancy and after treatment with exogenous steroids

Two experiments were performed to determine changes in the abundance of oestrogen and progesterone receptor (ER alpha and PR) mRNAs in equine endometrium during the oestrous cycle and early pregnancy, and under the influence of exogenous steroids. In Expt 1, endometrial biopsies were obtained from non-mated mares during oestrus and at days 5, 10 and 15 after ovulation, and from pregnant mares at days 10, 15 and 20 after ovulation. There were overall effects of day on the abundance of ER alpha (P = 0.0001) and PR (P = 0.0014) mRNAs. The amount of ER alpha mRNA decreased at day 10 of pregnancy, and PR mRNA was reduced at day 5 in non-mated mares and at day 15 of pregnancy, compared with oestrous values. Experiment 2 was conducted to determine the effects of exogenous steroids on endometrial ER alpha and PR mRNAs. Endometrial biopsies were obtained from 19 anoestrous mares that had been treated with vehicle, oestradiol, progesterone, or oestradiol followed by progesterone for either a short or a long duration. The steroid treatment affected the abundance of ER alpha mRNA (P = 0.0420), which was higher (P < 0.05) in the oestradiol group than in the group treated with oestradiol followed by long duration progesterone. The steroid treatment did not affect the abundance of PR mRNA. These results demonstrate that the amount of steroid receptor mRNA changes with the fluctuating steroid environment in the uterine endometrium of cyclic and early pregnant mares, and that the duration of progesterone dominance may affect ER alpha gene expression. In addition, factors other than steroids may regulate ER alpha and PR gene expression in equine uterine endometrium.     

αvβ3 Integrin and fibronectin expressions and their relation to estrogen and progesterone during placentation in swine

Mammalian pregnancy requires specific interactions between the conceptus and its mother that involve the endocrine system and adhesion molecules. The relation between adhesion molecules and their ligands at the fetal–maternal interface is crucial for developing a successful implantation. Progesterone (P4) and estrogen (E2) secreted by the porcine conceptus are required for the relation to be established. We investigated the expression of αvβ3 integrin and its ligand, fibronectin (FN), at the placental interface, and E2 and P4 concentrations in both serum and maternal and fetal placental extracts during placentation in swine. Placental and serum samples of crossbred sows at 17, 30, 60, 70, and 114 days gestation and no pregnant uteri were used. The presence of αvβ3 and FN were determined by immunohistochemistry, and E2 and P4 by chemiluminescence in homogenates of nonpregnant uterus (HoU), swine maternal placenta (HoPM), swine fetal placenta (HoPF) and serum. The expression of αvβ3 and FN increased at the interface at 17, 30 and 60 days gestation. Immunostaining decreased by 70 days. Serum E2 levels peaked at 17 days, then decreased, then increased again near term. The highest concentration of P4 occurred in HoPF at 70 days gestation, then decreased coincident with a decline in integrin and FN expression at the placental interface. High P4 levels during swine gestation may regulate the expression of αvβ3 integrin and FN at the placental interface for up to 70 days gestation. Other adhesion molecules and their ligands likely maintain the fetal–placental interface after 70 days.     

Supplementary corpora lutea monitoring allows progestin treatment interruption on day 70 of pregnancy in non-cyclic recipient mares

The present study evaluated the effect of altrenogest treatment during 70 or 120 days of gestation on pregnancy maintenance in non-cyclic recipient mares and correlated the hormonal interruption findings with number, supplementary corpora lutea (SCL) formation period, and plasma progesterone (P4). Twenty five mares were used as recipients during anestrus, transitional or ovulatory phase and were assigned into groups according to altrenogest treatment period (70ALT, 120ALT or Control groups) or reproductive status at beginning of treatment (Anestrus, Transition or Cyclic/Control groups). Mares were evaluated by ultrasonography and quantification of plasma progesterone to monitor pregnancy status, SCL formation and P4 profile. After hormonal withdrawal, abortion was only observed on group 70ALT. The days of first SCL formation were similar (p = 0.32) in the 70ALT and 120ALT groups and greater (p < 0.01) than the Control group. In addition, the first SCL formation period occurred later during gestation in the anestrus group than in the transitional or cyclic mares. Progesterone synthesis in non-cyclic mares occurred in more advanced gestational period and showed lower concentration during the 120 days in relation to cyclic mares. It is suggested that progestin treatment interruption in non-cyclic recipient mares at 70 days of gestation allows pregnancy maintenance when SCL are observed and at 120 days enables maintenance in all recipient mares. In addition, the first SCL development period occurs in different gestational phases during pregnancy among anestrus, transitional and cyclic mares. This study improves the understanding of pregnancy physiology and enables progestins treatment interruption on day 70 of pregnancy in non-cyclic pregnant recipient mares.     

Angiogenic activity of maternal and fetal placental tissues of ewes throughout gestation

In Study 1, explants of caruncular and intercaruncular endometrium and fetal membrane were collected from ewes (5-6/day) on Days 11-13, 16-18 and 21-23 after mating and Days 10-12 after oestrus, and incubated for 24 h. Explant-conditioned media were evaluated for their effects on endothelial cell proliferation. Both caruncular and intercaruncular endometrium secreted factor(s) which stimulated endothelial cell proliferation, and which appeared to be greater than 100 x 10(3) Mr and heat-labile. In Study 2, conditioned media from explant incubations of caruncular and intercaruncular endometrium, cotyledon and intercotyledonary fetal membrane obtained from ewes (6-7/day) on Days 40, 65, 90, 115 and 140 after mating were evaluated for their effects on endothelial cell proliferation. Caruncular and intercaruncular endometrium and intercotyledonary fetal membrane secreted factor(s) which inhibited endothelial cell proliferation. Media from cotyledonary explants tended to stimulate endothelial cell proliferation on Day 115. Conditioned media from cotyledonary explants obtained from 3 additional ewes at Day 120 of gestation stimulated endothelial cell proliferation, and this activity also appeared to be greater than 100 x 10(3) Mr. Placental angiogenesis in ewes therefore appears to be modulated by both maternal and fetal placental tissues via stimulatory and inhibitory factors.     

Maternal age and parity influence ultrasonographic measurements of fetal growth in Dutch Warmblood mares

Ultrasonographic examination of the equine fetus in mid-late gestation is usually performed only if there are concerns about fetal or maternal health. Even then it is difficult to determine whether development is ‘normal’ for gestational age because the reference values include considerable error margins. This study examined maternal factors that influence fetal growth with the aim of producing more precise late gestation fetal growth curves for Dutch Warmblood horses. Fetal development was monitored at 2-week intervals from day 100 of gestation until term in 32 mares ranging from 4 to 18 years in age; seven of the mares were primiparous. Transrectal and/or transabdominal ultrasonographic measurement of the fetal eye orbit, cranium, aorta, heart rate and of the combined thickness of uterus and placenta (CTUP) were performed using a portable ultrasound machine equipped with 6 MHz linear and 3.5 MHz curved array probes.During days 100–250 of gestation, the CTUP was thicker in primiparous than multiparous mares (p < 0.05). After day 220 the maximum cross-sectional area, but not diameter, of both the eye orbit and cranium were also greater in primiparous than multiparous mares (p < 0.05). Fetal aorta diameter was not influenced by parity but was affected by maternal age, being smaller in mares ≥15 years of age than younger animals (p < 0.05). Only biparietal cross-sectional surface area and aorta diameter increased linearly throughout late gestation. However, even allowing for the effects of parity and maternal age, the late gestational variation in fetal size is such that serial measurements may be required to definitively identify abnormal development.     

Plasma progestogen levels in pregnant mares following administration of exogenous progesterone

Twelve pregnant mares were used in a switchback design with two groups of six mares each. On day 255 of gestation, group A was administered repository progesterone (250 mg, IM) and group B progesterone in sesame oil (250 mg, IM). Jugular vein plasma was taken 15 min pre-injection and post-injection at 30 min, 1, 3, 6, 9, 12, and 24 hours. Additional samples were taken on days 2, 3, 4, 6, and 8. Both groups were then assigned to the opposite treatment on day 270 of gestation. Concentrations of plasma progestogens after injection with progesterone in sesame oil were not different (P>.05) from values obtained using repository progesterone through the 8 days studied. Mean progestogen concentrations after injection of progesterone in sesame oil were, however, consistently higher through 48 hr than those given repository progesterone. Concentrations of progestogens were not different (P>.05) from pre-injection concentrations by day 6 post-injection using either vehicle.     

The effects of gestational age and intrafetal ACTH administration on the concentration of progesterone in the fetal membranes, endometrium, and myometrium of pregnant sheep

To examine the hypothesis that progesterone withdrawal from intrauterine tissues is a prerequisite to spontaneous labour or labour induced by administering ACTH to the ovine fetus, we measured the concentration of progesterone in amnion, chorion, endometrium, and myometrium of sheep at different stages of pregnancy and during ACTH-induced labour. There was no significant change in the concentration of progesterone nor in the progesterone:estradiol ratio in amnion or chorion in association with either spontaneous or ACTH-induced labour. The concentration of progesterone in endometrium rose significantly between days 50-60 and days 130-135 of gestation and decreased at term. There was also a fall in the progesterone:estradiol ratio in endometrium between days 130-135 and term. Neither the progesterone concentration not the progesterone:estradiol ratio changed in endometrium during ACTH-induced labour. In the myometrium the concentration of progesterone rose significantly between days 50-60 and day 100 of pregnancy and decreased between day 100 and days 130-135, with a further decline towards term. After intrafetal ACTH there was no change in the concentration of progesterone in the myometrium, although there was a fall in the progesterone:estradiol ratio. We conclude that labour occurring spontaneously at term is associated with a decrease in the progesterone concentration of maternal intrauterine tissues, the myometrium and endometrium. In contrast, there is no decline in the progesterone concentrations of the fetal membranes, the amnion and chorion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ontogeny of uteroplacental progestagen production in pregnant mares during the second half of gestation

In pregnant mares during late gestation, little, if any, progesterone (P4) is found in the maternal circulation. Hence, quiescence of the equine uterus is believed to be maintained by metabolites of pregnenolone and P4 known as progestagens, which are produced by the uteroplacental tissues. However, little is known about the ontogeny, distribution, or actual rates of uteroplacental progestagen production in pregnant mares and their fetuses during the second half of pregnancy. Therefore, the present study measured the rates of uteroplacental uptake and output of eight specific progestagens in chronically catheterized, pregnant pony mares from 180 days to term. No significant uteroplacental uptake of any of the eight individual progestagens was observed from the uterine circulation. In contrast, significant uteroplacental uptake was observed for five of the eight individual progestagens from the umbilical circulation, and the uptakes increased toward term. The major uteroplacental progestagen outputs were 5 alpha-pregnane-3,20-dione (5 alphaDHP) and 20 alpha-hydroxy-5 alpha-pregnan-3-one (20 alpha 5P). These were released into both the umbilical and uterine circulations at rates that increased toward term. The majority of the total uteroplacental 20 alpha 5P output was distributed into the uterine circulation at all gestational ages studied. In contrast, distribution of the total uteroplacental 5 alphaDHP output switched from preferential delivery into the uterine circulation before 220 days of gestation to release predominantly into the umbilical circulation after 260 days. These findings demonstrate that uteroplacental progestagen production changes during the second half of gestation, which may have important implications for the maintenance of pregnancy and the onset of labor in the mare.     

Treatments resulting in pregnancy in nonovulating, hormone-treated oocyte recipient mares

Synchronization of follicle growth between oocyte donor and recipient mares is difficult. To avoid this, recipient mares in a clinical program were used during a period of low follicular activity, and were treated with estrogen before transfer and progesterone after transfer. Five pregnancies were established after oocyte transfer to nonovulating, hormone-treated recipient mares. One pregnancy was lost before 30 d gestation, and the other 4 foals were carried to term. One foal died at birth. Establishment and maintenance of pregnancy in these mares indicates that nonovulating, hormone-treated mares may offer an alternative to cyclic recipients in oocyte transfer programs.     

Cortisol and progestin release, heart rate and heart rate variability in the pregnant and postpartum mare, fetus and newborn foal

The mechanisms leading to parturition in the horse in many aspects differ from those in other species. Pregnancy is maintained not by progesterone but by 5α-pregnanes and the progestin precursor pregnenolone originates from the fetus. As parturition approaches, the fetal adrenal switches from pregnenolone to cortisol synthesis but it is not known whether cortisol crosses the placenta. We hypothesized that in parallel to fetal cortisol release, cortisol in the maternal circulation increases before foaling and this increase can be determined in both saliva and plasma. In addition, maternal, fetal and neonatal heart rate and heart rate variability were measured. In 25 pregnant mares, saliva for cortisol analysis was collected 4 times daily from 15 days before to 5 days after foaling. In 13 mares, in addition, fetomaternal electrocardiogram (ECG) recordings were made and blood samples for progestin and cortisol analysis were collected once daily. Heart rate (HR) was recorded until 5 days after foaling. The heart rate variability (HRV) variables standard deviation of the beat-to-beat (RR) interval (SDRR) and root mean square of successive RR differences (RMSSD) were calculated. From Days 15 to 4 before parturition, progestin concentration increased (peak 267 ± 42 ng/mL) and decreased thereafter (P < 0.05, day of foaling 113 ± 18 ng/mL). A prepartum increase in maternal cortisol concentrations was evident in blood (P < 0.05) and saliva (P < 0.05) and paralleled the decrease in progestin concentrations. In mares, HR remained constant during the last days of pregnancy but decreased within one day after parturition (P < 0.05) while maternal HRV did not change. In the fetus and neonate, HR increased from before to after birth (P < 0.05) indicating increasing demands on the cardiovascular system with adaptation to extrauterine life.     

Effect of exogenous gonadal steroids and pregnancy on uterine luminal prostaglandin F in mares

Two experiments were conducted to assess the effect of exogenous hormone treatment on uterine luminal prostaglandin F (PGF). In the first experiment ovariectomized pony mares received either corn oil (21 days, n = 3), estradiol valerate (21 days, n = 3), progesterone (21 days, n = 3) or estradiol valerate (7 days) followed by progesterone (14 days, n = 4). Progesterone treated mares had higher (P<.01) uterine luminal PGF compared with all other groups, and no differences were detected between other treatment comparisons. In Experiment II, uterine fluid was collected from 4 ovariectomized horse mares before and after treatment with estradiol valerate (7 days) followed by progesterone (50 days). Pretreatment uterine luminal PGF levels were lower (P<.001) than post-treatment levels (.03 vs 76.80 ng/ml). In a third experiment PGF was measured in uterine fluid of pony mares on days 8, 12, 14, 16, 18 and 20 of the estrous cycle and pregnancy. In nonpregnant mares a day effect P<.03) was observed in which uterine fluid PGF increased during the late luteal phase and declined thereafter. In contrast, no day effect was observed in pregnant animals and uterine luminal PGF was lower (P<.001) than in cycling animals. These studies indicate that exogenous progesterone administration results in a large increase in uterine luminal PGF, whereas, pregnancy results in suppression. Taken collectively with previous work from our laboratory, these results suggest that while the endometrium of pregnant mares is capable of producing large amounts of PGF, the presence of a conceptus impedes its synthesis and/or release which allows for luteal maintenance.     

Establishment and maintenance of pregnancy after embryo transfer in ovariectomized mares treated with progesterone

Pregnancy was established and maintained after embryo transfer in 3 ovariectomized mares treated with progesterone only. Four ovariectomized mares were used as recipients, and 7 transfers were performed. Progesterone in oil, 300 mg i.m. daily, was given starting 5 days before transfer of a 7-day embryo. If the mare was pregnant at 20 days, progesterone treatment was continued to 100 days of gestation. The 3 pregnant mares carried to term and delivered live foals with normal parturition, lactation and maternal behaviour. No differences were seen between pregnant and non-pregnant ovariectomized mares in jugular plasma concentrations of oestrogen, LH or FSH from day of transfer (Day 7) to Day 20. Pregnant ovariectomized mares showed a rise in LH, reflecting production of horse CG, starting at Day 36. Oestrogen values remained low until Day 50.     

Effects of bromocriptine and perphenazine on prolactin and progesterone concentrations in pregnant pony mares during late gestation

Pregnant pony mares in Group A (n = 4) received i.m. injections at 07:00 and 17:00 h of 0.8 mg bromocriptine/kg body weight 0.75 per day beginning on Day 295 of gestation and continuing until parturition. Group B (n = 4) was treated similarly, but perphenazine was administered orally at 0.375 mg/kg body weight twice a day beginning on Day 305 of gestation and continuing until parturition. Mares in Group C (n = 3) received i.m. injections of saline. Mean plasma prolactin and progesterone concentrations were greater (P less than 0.05) for mares in Group C than in Groups A and B from 295 to 309 days of gestation. From 305 days of gestation, plasma prolactin and progesterone concentrations were greater (P less than 0.05) in Group B and C than in Group A mares. Progesterone and prolactin concentrations increased over this period for Group B and Group C mares, but remained constant in Group A mares. From 10 days pre partum through foaling, mares in Group A had lower progesterone (P less than 0.05) and prolactin (P less than 0.01) concentrations than Group B and C mares. All mares in Group A were agalactic at foaling, while all mares in Groups B and C had normal milk secretion. Gestation was longer (P less than 0.05) in Group A than in Group C mares. In Group A, 2 mares retained the placenta for greater than 3 h, 3 mares had dystocia and all 4 mares had thickened, haemorrhagic placentae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhanced or Reduced Fetal Growth Induced by Embryo Transfer into Smaller or Larger Breeds Alters Post-Natal Growth and Metabolism in Pre-Weaning Horses

In equids, placentation is diffuse and nutrient supply to the fetus is determined by uterine size. This correlates with maternal size and affects intra-uterine development and subsequent post-natal growth, as well as insulin sensitivity in the newborn. Long-term effects remain to be described. In this study, fetal growth was enhanced or restricted through ET using pony (P), saddlebred (S) and draft (D) horses. Control P-P (n = 21) and S-S (n = 28) pregnancies were obtained by AI. Enhanced and restricted pregnancies were obtained by transferring P or S embryos into D mares (P-D, n = 6 and S-D, n = 8) or S embryos into P mares (S-P, n = 6), respectively. Control and experimental foals were raised by their dams and recipient mothers, respectively. Weight gain, growth hormones and glucose homeostasis were investigated in the foals from birth to weaning. Fetal growth was enhanced in P-D and these foals remained consistently heavier, with reduced T₃ concentrations until weaning compared to P-P. P-D had lower fasting glucose from days 30 to 200 and higher insulin secretion than P-P after IVGTT on day 3. Euglycemic clamps in the immediate post-weaning period revealed no difference in insulin sensitivity between P-D and P-P. Fetal growth was restricted in S-P and these foals remained consistently lighter until weaning compared to S-D, with elevated T₃ concentrations in the newborn compared to S-S. S-P exhibited higher fasting glycemia than S-S and S-D from days 30 to 200. They had higher maximum increment in plasma glucose than S-D after IVGTT on day 3 and clamps on day 200 demonstrated higher insulin sensitivity compared to S-D. Neither the restricted nor the enhanced fetal environment affected IGF-1 concentrations. Thus, enhanced and restricted fetal and post-natal environments had combined effects that persisted until weaning. They induced different adaptive responses in post-natal glucose metabolism: an early insulin-resistance was induced in enhanced P-D, while S-P developed increased insulin sensitivity.     

Plasma concentrations of pregnancy-associated glycoprotein-1 (PAG-1) in high producing dairy cows suffering early fetal loss during the warm season

The present study was designed to establish whether plasma pregnancy-associated glycoprotein-1 (PAG-1) measurements during the early fetal period can be associated with early fetal loss. Blood samples were obtained and ultrasound controls performed on days 35, 42, 49, 56, and 63 of gestation or until pregnancy loss from 98 lactating dairy cows. Radioimmunoassay systems were used to determine PAG-1 and progesterone concentrations. Of the 98 pregnancies investigated 18 (18.4%) suffered early fetal loss: 15 (18.5%) in cows carrying singletons, and 3 (16.7%) in twin pregnancies. In cows suffering pregnancy loss, all living embryos registered on day 35 seemed normal in size and development in all weekly ultrasound controls before fetal expulsion. Using analysis of variance, plasma PAG-1 and progesterone values were not different between no loss and fetal loss groups for every gestation period. Based on the odds ratio, and considering only PAG-1 values obtained on day 35 of gestation, the risk of fetal loss was 10 and 6.8 times more likely in cows with low (<2.5 ng/ml) and high (>4 ng/ml) PAG-1 values, respectively, than in cows with medium PAG-1 values, used as reference. Of the 10 inseminating bulls included in the study, one was related to increased fetal loss by odds ratio of 21.7, whereas one bull was attributed fetal loss rate reduced by odds ratio of 12.5 (1/0.08) These findings can have a clear clinical application: PAG-1 measurements from one single sample taken on day 35 of gestation provided more useful information than a series of values obtained from day 35 to 63 of gestation, and can be indicators of subsequent fetal loss.