In mammalian pregnancy, maternal cardiovascular adaptations must match the requirements of the growing fetus(es), and respond to physiologic and pathologic conditions. Such adaptations are particularly demanding for mammals bearing large-litter pregnancies, with their inherent conflict between the interests of each individual fetus and the welfare of the entire progeny. The mouse is the most common animal model used to study development and genetics, as well as pregnancy-related diseases. Previous studies suggested that in mice, maternal blood flow to the placentas occurs via a single arterial uterine loop generated by arterial-arterial anastomosis of the uterine artery to the uterine branch of the ovarian artery, resulting in counter bi-directional blood flow. However, we provide here experimental evidence that each placenta is actually supplied by two distinct arterial inputs stemming from the uterine artery and from the uterine branch of the ovarian artery, with position-dependent contribution of flow from each source. Moreover, we report significant positional- and inter-fetal dependent alteration of placental perfusion, which were detected by in vivo MRI and fluorescence imaging. Maternal blood flow to the placentas was dependent on litter size and was attenuated for placentas located centrally along the uterine horn. Distinctive apposing, inter-fetal hemodynamic effects of either reduced or elevated maternal blood flow, were measured for placenta of normal fetuses that are positioned adjacent to either pathological, or to hypovascular Akt1-deficient placentas, respectively. The results reported here underscore the critical importance of confounding local and systemic in utero effects on phenotype presentation, in general and in the setting of genetically modified mice. The unique robustness and plasticity of the uterine vasculature architecture, as reported in this study, can explain the ability to accommodate varying litter sizes, sustain large-litter pregnancies and overcome pathologic challenges. Remarkably, the dual arterial supply is evolutionary conserved in mammals bearing a single offspring, including primates.

Noninvasive imaging is a critical part of the study of developing embryos/fetuses, particularly in the context of alterations of gene expression in genetically modified animals. However, in litter-bearing animals, such as mice, the inability to accurately identify individual embryo/fetus in utero is a major obstacle to longitudinal, noninvasive in vivo studies. Arterial spin labeling MRI was adopted here to determine the fetal order along the uterine horns in vivo, based on the specific pattern of dual arterial blood supply within the mouse uterine horns. Blood enters the mouse uterus cranially through the ovarian artery and caudally through the uterine artery. Saturation slices were alternately placed on the maternal heart or on the bifurcation point of the common iliac artery, thereby saturating either downward inflow via the ovarian arteries or upward inflow via the uterine arteries, respectively. Saturation maps provided a unique signature with highly significant correlation between the direction-dependent magnetization transfer and the position of the fetuses/placentas along the uterine horns. The bidirectional arterial spin labeling-MRI method reported here opens possibilities to determine and pursue phenotypic alterations in fetuses and placentas in longitudinal studies of transgenic and knockout mice models, and for studying defects in placental vascular architecture.

Reliable methods for regulating oestrus and superovulation in equine embryo transfer (ET) programs are desirable. The objective in this study was to compare two oestrus synchronization methods combined with equine follicle-stimulating hormone (eFSH) treatment in an ET program. In the progesterone and estradiol-17β (P&E) group, mares (n=12) were given progesterone and estradiol-17β, daily for 10 days, followed by prostaglandin (PG)F(2α) on the last day. In the PG group, mares (n=12) were given PGF(2α) 5 days post-ovulation. In both groups donor mares were allocated to eFSH therapy, and were subsequently bred. Embryo recovery and transfer were performed routinely. The interval to ovulation (mean ± SEM, range) was not statistically different between donor mares in the P&E group (10.2±0.3, 9-12 days) and donor mares in the PG group (8.7±0.7, 4-12 days). Among donor mares, the synchrony of ovulations was higher following the P&E regimen (P<0.05); however, there was a tendency (P<0.06) for fewer ovulations than in the PG group (1.5±0.3 vs. 2.5±0.4 ovulations, respectively). Embryo recovery (0.9±0.3 vs. 1.4±0.3 embryo/recovery) and recipient pregnancy rate per transferred embryo (4/9, 44% vs. 4/15, 27%) were similar. It was concluded that the P&E regimen was more reliable for synchronization of oestrus in eFSH-treated mares but the fewer ovulations may curtail any advantage of this regimen.

BACKGROUND: Induction of multiple ovulations, or superovulation, may potentially increase the efficiency of equine embryo transfer programs. Our objective was to investigate the effects of equine follicle-stimulating hormone (eFSH) treatment on the success rate of embryo transfer programs in mares. METHODS: In the research facility of the University of Saskatchewan, Canada, we studied 12 donor mares and 37 recipient mares during the physiological breeding season. Donor mares were used in two consecutive oestrous cycles: the first served as the control cycle and in the second an eFSH regimen was applied (eFSH cycle). In the control cycle, mares were administered human chorionic gonadotropin (hCG) to induce ovulation when a follicle ≥35 mm in diameter was detected by transrectal ultrasonographic examination. In the second oestrous cycle, twice-daily eFSH treatment was initiated when a follicle ≥25 mm was detected and treatment ceased when a follicle ≥35 mm was present, at which time hCG was administered. All donor mares were artificially inseminated while in oestrus using fresh semen collected from a stallion of proven fertility. At 8 days post-ovulation, embryos were recovered transcervically and transferred individually to the uterus of a synchronised recipient mare. RESULTS: The eFSH treatment stimulated the ovary and resulted in greater numbers of ovulations and recovered embryos; however the recovered embryos tended to have a lower morphological grade than the control embryos, and the recipient pregnancy rate per transferred embryo was lower than anticipated. CONCLUSION: The numbers of recipient pregnancies and foals born that resulted from eFSH treatment were not different from the control.

The objective of this study was to compare the efficacy of purified equine- and porcine-FSH treatment regimes in mares in early vernal transition. Mares (n = 22) kept under ambient light were examined ultrasonographically per-rectum, starting January 30th. They were assigned to one of two treatment groups using a sequential alternating treatment design when a follicle >or= 25 mm was detected. In the eFSH group, mares were treated twice daily with equine-FSH, and in the pFSH group mares were treated twice daily with porcine-FSH; treatments were continued until follicle(s) >or= 35 mm, and 24 h later hCG was administered. Oestrous mares were inseminated with fresh semen and examined for pregnancy on days 11-20 post-ovulation. In the eFSH group, 11/11 (100%) mares developed follicle(s) >or= 35 mm, 8/11 (73%) ovulated and 6/8 (75%) conceived. In the pFSH group, 5/11 (45%) developed follicle(s) >or= 35 mm, 4/11 (36%) ovulated and 3/4 (75%) conceived. Treatment with eFSH resulted in a greater ovarian stimulation; higher number of pre-ovulatory-sized follicles, higher number of ovulations and higher number of embryos (p < 0.05). Following ovulation, serum progesterone concentrations were correlated with the number of CLs and supported early embryonic development; maternal recognition of pregnancy occurred in all pregnant mares. We concluded that eFSH can be used to effectively induce follicular growth and ovulation in vernal transitional mares; however, if bred, diagnosis and management of twins' pregnancies would be required prior to day 16 because of the increased risk of multiple embryos per pregnancy. Conversely, the current pFSH treatment regime cannot be recommended.

A 5-year-old Arabian stallion was managed for breeding with fresh/extended semen during a period of 8 months with a resulting per cycle pregnancy rate of 26.3%. The stallion was in good health and no abnormalities of the reproductive tract were observed. Evaluation of several ejaculates revealed that sperm production and semen quality were mostly unchanged during the period of evaluation, that sperm production was normal and that semen quality was extremely poor. The most prevalent sperm defects were abnormal heads and mid-pieces. Most abnormal heads were microcephalic and/or tapered and considerable variation in sperm head dimensions within and among ejaculates was observed. A unique defect characterized by swollen/roughened mid-piece caused by accumulation of cytoplasmic-like material and abnormal mitochondrial sheath was observed. Nuclear vacuoles, acrosome defects, and teratoids were also prevalent and most sperm presented multiple abnormalities. The absence of any clear cause or any signs of testicular degeneration, combined with normal sperm production, and constant abnormal sperm production suggest an inherent, congenital disturbance of spermatogenesis as the cause of teratospermia in this case.

The objective was to compare the effects of eFSH and deslorelin treatment regimes on ovarian stimulation and embryo production of donor mares in early spring transition. Starting January 30th, mares kept under ambient light were examined by transrectal ultrasonography. When a follicle > or =25 mm was detected, mares were assigned to one of two treatment groups, using a sequential alternating treatment design. In the eFSH group, mares (n=18) were treated twice daily with eFSH (12.5mg im) until they achieved a follicle > or =35 mm; hCG was given 36 h later. In the deslorelin group, mares (n=18) were treated twice daily with deslorelin (63 microg im) until a follicle > or =35 mm was detected, and then they were given hCG. Estrous mares were inseminated with fresh semen. Eight days after ovulation, embryo recovery attempts were performed. In each group, 14/18 (78%) mares ovulated following the eFSH or deslorelin treatment regimes. The mean (95% CI) interval from treatment initiation to ovulation was 8.2d (7.3, 8.9) and 7.2d (6.2, 8.1) in the eFSH and deslorelin groups, respectively. In the eFSH group, the number of ovulations was significantly higher (mean+/-S.E.M.; 3.4+/-0.4 vs. 1.1+/-0.1 ovulations), and more embryos were recovered (2.6+/-0.5 vs. 0.4+/-0.2 embryos/recovery attempt). We concluded that eFSH and deslorelin treatment regimes were equally effective in inducing ovulation in early transitional mares, within a predictable time of treatment; however, the eFSH regime increased the number of ovulations and embryos recovered per mare.

Superovulatory treatment may potentially increase the embryo recovery rate and the per-cycle pregnancy rate in normal or subfertile mares that are managed properly. However, some studies suggest a possible negative effect of superovulatory treatment on ovarian follicular maturation and embryo viability. Objectives of the present study were to investigate the early effects of eFSH treatment in reproductively normal mares in terms of: folliculogenesis, pregnancy rate, early embryonic development, reproductive tract parameters (tone and edema), and serum estradiol-17beta and progesterone concentrations. Reproductively sound mares (n=26) were evaluated daily by transrectal palpation and ultrasonography. Five days after spontaneous ovulation, mares were randomly assigned to one of two treatment groups. In the eFSH group, mares (n=16 estrous cycles) were administered eFSH twice daily; beginning when a follicle > or =20mm was detected, and continuing until at least one follicle reached a diameter of > or =35 mm. PGF2alpha was administered 2 days following initiation of eFSH therapy, and hCG was administered approximately 36h after cessation of eFSH therapy. In the control group, mares (n=26 estrous cycles) were administered PGF2alpha 7 days after spontaneous ovulation, and hCG when a follicle > or =35 mm was detected. All mares were bred with fresh semen, monitored for ovulation (Day 0), and evaluated for pregnancy on Days 11-16. Serum estradiol-17beta and progesterone concentrations were analyzed using radioimmunoassay on the Day of hCG administration, and Days 8, 11 and 16. Mares treated with eFSH had more follicles > or =30 mm at the time of hCG administration (2.6+/-0.4 compared with 1.1+/-0.1; P<0.01), and more ovulations (2.3+/-0.5 compared with 1.1+/-0.3; P<0.01). However, pregnancy rates were not significantly different between groups (50%; 8/16 compared with 62%; 16/26). Mean overall daily growth rate of embryonic vesicles from Day 11 to 16 was not statistically different between the two groups (3.3+/-0.3 compared with 3.7+/-0.1 mm/day) (P=0.2); however, was more variable (P<0.01) in the eFSH group (95%CI: 2.6-3.8mm/day) than in the control group (95%CI: 3.5-3.9 mm/day). Administration of eFSH modified the reproductive tract variables and serum concentrations of progesterone and estradiol-17beta on the days that oocyte maturation, fertilization, and early embryonic development are expected to occur. These alterations may be related to the greater incidence of non-ovulatory follicles (25% compared with 0%), fewer embryos per ovulation rate (0.3+/-0.1 compared with 0.6+/-0.1), and the lesser than expected pregnancy rates in the eFSH-treated mares.

Reliable methods of regulating estrus and stimulating superovulations in equine embryo transfer programs are desirable. Our objectives were to investigate the efficacy of a progesterone and estradiol-17beta (P&E) estrus synchronization regimen in mares with and without subsequent equine follicle-stimulating hormone (eFSH) treatment and to examine the effects of eFSH on folliculogenesis and embryo production. Cycling mares were treated with P&E daily for 10 d. On the final P&E treatment day, prostaglandin F(2alpha) was administered, and mares were randomly assigned to one of two treatment groups (n=20 mares/group). In both groups, mares were examined daily by transrectal ultrasonography. In the eFSH group, twice-daily eFSH treatments were initiated at follicle diameter 20 to 25 mm and ceased at follicle > or =35 mm; human chorionic gonadotrophin (hCG) was administered after 36 h. In the control group, eFSH treatments were not given, but hCG was administered at follicle > or =35 mm. Mares were inseminated with fresh semen, and embryo recovery attempts were performed 8 d postovulation. Synchrony of ovulations within each group appeared to be similar. Six mares in the eFSH group failed to ovulate. The eFSH treatment resulted in higher (P<0.05) numbers of preovulatory follicles and ovulations; however, embryo recovery rate did not increase (eFSH 1.0+/-0.4 vs. control 0.95+/-0.1 embryos/recovery attempt), and embryo per ovulation rate was significantly lower (36% vs. 73%). The eFSH-treated mares had significantly higher frequency of nonovulatory follicles (28% vs. 0) and higher periovulatory serum concentrations of estradiol-17beta. Based on our findings, combined P&E and eFSH regimens cannot be recommended for cycling donor mares.

The objective was to compare the reproductive performances associated with the first (Cycle-1), second (Cycle-2), and mid-season (MS-Cycle) ovulations of the breeding season in donor mares that were treated with equine-FSH (eFSH) in the early vernal transition. Mares (n=15) kept under ambient light were examined ultrasonographically per-rectum starting January 30. When an ovarian follicle > or =25mm in diameter was detected, twice daily eFSH treatments were initiated. The eFSH treatments ceased when a follicle > or =35mm was detected, and 36h later hCG was administered. Thereafter, mares were artificially inseminated every 48h until ovulation (Day 0). Trans-cervical embryo recovery attempts were performed on Day 8, and subsequently PGF2alpha was administered. Equine FSH was not administered in the subsequent estrous cycles. In Cycle-2 and in the MS-Cycle, hCG was administered when a follicle > or =35mm was detected; breeding, embryo recovery, and PGF2alpha administration, were similar to Cycle-1. Mares had an untreated estrous cycle (no treatment or breeding) between Cycle-2 and the MS-Cycle. All mares developed follicle(s) > or =35mm after 4.9+/-0.6 days of eFSH treatment, and subsequently ovulations occurred; mean (95% CI) interval from treatment initiation to ovulation was 7.9 (6.5-9.3) days. The number of preovulatory follicles (> or =30mm) at the time of hCG administration (Cycle-1: 2.2+/-0.3 compared with Cycle-2: 1.0+/-0 compared with MS-Cycle: 1.1+/-0.1 follicles), and the number of ovulations (2.5+/-0.4 compared with 1.0+/-0 compared with 1.1+/-0.1 ovulations) were greater (p<0.05) in Cycle-1. Nevertheless, mean embryo numbers did not differ among cycles (0.8+/-0.2 compared with 0.5+/-0.1 compared with 0.5+/-0.1 embryo/mare). On average, embryo morphology grade was less (p<0.05) in Cycle-1 as compared to non-eFSH cycles (combined Cycle-2 and MS-Cycle). This impaired embryo quality could be due to a seasonal effect, or negative effect of the eFSH treatment, which was possibly related to alterations in the hormonal environment (estradiol-17beta and progesterone). A prolonged IOI (>21 days) was recorded in 7 of 15 mares following the Cycle-1 ovulation, but not subsequently. In conclusion, eFSH treatment of vernal transitional donor mares stimulated ovulation within only few days of treatment, and the following embryo recovery rate was at least as good as in the subsequent estrous cycles; however, on average, embryos were morphologically impaired. In subsequent estrous cycles in the breeding season, ovulations, embryo recovery rates, and embryo variables did not appear to be negatively affected; however, the first inter-ovulatory interval of the breeding season was prolonged in approximately half of the mares.