T. W. Geary

Follicular determinants of pregnancy establishment and maintenance

Synchronization of dominant follicle development and control of ovulation/oocyte retrieval are commonly used assisted reproductive technologies in both cattle and humans. The final maturation of the dominant follicle is intimately tied to the final maturation of the oocyte, preovulatory secretion of estradiol, preparation of follicular cells for luteinization, postovulatory secretion of progesterone and endocrine control of the oviductal and uterine environment for gamete and embryo development. The physiological maturity of a dominant/ovulatory follicle can affect the establishment and maintenance of pregnancy. Premature induction of the ovulatory process can reduce pregnancy rates and increase late embryonic/fetal mortality in cattle, which is likely mediated through inadequate oocyte competence and a compromised maternal environment. Oocyte competence increases with follicular maturity and is dependent upon acquisition of a complete complement of mRNA transcripts and establishment of the appropriate epigenetic marking of the oocyte genome before the preovulatory gonadotropin surge. Preovulatory secretion of estradiol is a reflection of follicular maturity and affects the oocyte, follicular cells, oviduct and uterus. The corpus luteum is a continuation of follicular maturation and rate of progesterone secretion following ovulation is linked to fertility. Advancements in our understanding of how the follicular microenvironment affects pregnancy establishment and maintenance will improve the efficiency of assisted reproductive technologies in all species. The purpose of this review is to discuss how follicular microenvironment, oocyte competence, preovulatory secretion of estradiol and postovulatory secretion of progesterone can affect pregnancy establishment and embryo/fetal survival, with an emphasis on cattle.

Reproductive performance of heifers offered ad libitum or restricted access to feed for a one hundred forty-day period after weaning.

Reproductive performance was evaluated in composite heifers born over a 3-yr period that were randomly assigned to control (fed to appetite; n = 205) or restricted (fed at 80% of that consumed by controls adjusted to a common BW basis; n = 192) feeding for a 140-d period, beginning about 2 mo after weaning at 6 mo of age and ending at about 12.5 mo of age. Heifers were fed a diet of 67% corn silage, 18% alfalfa, and 9% of a protein-mineral supplement (DM basis). Restricted heifers consumed 27% less feed over the 140 d and had less ADG (0.53 +/- 0.01 vs. 0.65 +/- 0.01 kg/d; P < 0.001) than control heifers. After 140 d, all heifers were placed in common pens and subjected to an estrous synchronization protocol to facilitate AI at about 14 mo of age. Heifers were then exposed to bulls for the remainder of a 51-d breeding season. Average BW of heifers diverged within 28-d after initiation of feed restriction, and differences (P < 0.001) persisted through the prebreeding period (309 +/- 1 vs. 326 +/- 1 kg at approximately 13.5 mo of age) and subsequent grazing season (410 +/- 2 vs. 418 +/- 2 kg at about 19.5 mo of age). From the end of the 140-d restriction at about 12.5 to 19.5 mo of age, ADG was greater (P < 0.001) in restricted heifers than control heifers (0.51 +/- 0.01 vs. 0.47 +/- 0.01 kg/d). Proportion of heifers attaining puberty by 14 mo of age tended to be less (P = 0.1) in restricted (60 +/- 3%) than control-fed heifers (68 +/- 3%). Mean BW at puberty was less (P < 0.01) in restricted (309 kg) than control (327 kg) heifers. Pregnancy rate from AI tended to be less (P = 0.08) in restricted (48 +/- 4%) than control heifers (57 +/- 3%). Proportion of animals that were pubertal at breeding and pregnant from AI were positively associated (P < 0.1) with heifer age and ADG from birth to beginning of study. Final pregnancy rates were 87 and 91% for restricted and control heifers, respectively (P = 0.27). Day of breeding season that conception occurred was negatively associated with ADG from birth to weaning (P = 0.005), but was not associated with ADG within treatment (P = 0.60). Economic analysis revealed a

Preovulatory estradiol and the establishment and maintenance of pregnancy in suckled beef cows.

33 reduction in cost to produce a pregnant heifer under the restricted protocol when accounting for pregnancy rates and differences in BW and market prices between selection at weaning and marketing as open heifers at l.5 yr of age. A potential economic advantage exists for rearing replacement heifers on a restricted level of feeding during the postweaning period.

TRIENNIAL REPRODUCTION SYMPOSIUM: Deficiencies in the uterine environment and failure to support embryonic development

In postpartum beef cows, GnRH-induced ovulation of small dominant follicles decreased pregnancy rates and increased late embryonic/fetal mortality. In Exp. 1, single ovulation reciprocal embryo transfer (ET) was used to examine the relationship between preovulatory serum concentrations of estradiol at GnRH-induced ovulation in donor and recipient cows and establishment and maintenance of pregnancy. Suckled beef cows (n = 1,164) were administered GnRH (GnRH1, 100 μg) on d -9 (GnRH1), PGF(2α) on d -2, and GnRH2 (GnRH2, 100 μg) on d 0 (CO-Synch protocol) either with (donors; n = 810) or without (recipients; n = 354) AI. Single embryos (n = 394) or oocytes (n = 45) were recovered from the donor cows (d 7; ET) and all live embryos were transferred into recipients. Serum concentration of estradiol at GnRH2 was positively correlated with follicle size at GnRH2 (r = 0.45, P < 0.01) and progesterone at ET (r =0.34, P < 0.01). Donor cows with greater estradiol at GnRH2 were more likely to yield an embryo than an unfertilized oocyte (P < 0.01). Donor and recipient cows were retrospectively divided into 4 groups [low estradiol (<8.4 pg/mL) or high estradiol (≥8.4 pg/mL)] based on serum concentration of estradiol at GnRH2. Pregnancy rate at d 27 for low-low (n = 78), low-high (n = 80), high-low (n = 91), and high-high (n = 101) groups (donor-recipient, respectively) was 45, 65, 43, and 61% respectively (P < 0.02). Because recipient cows with greater estradiol concentration at GnRH2 had greater pregnancy rates in Exp. 1, the objective of Exp. 2 was to evaluate the effect of estradiol supplementation on pregnancy rate. Ovulation was synchronized in suckled beef cows (n = 600) using the CO-Synch protocol with the insertion of a controlled internal drug release (CIDR; intravaginal progesterone supplement) from d -9 until d -2. Approximately one-half of the cows (n = 297) received an injection of estradiol cypionate (ECP; 0.5 mg intramuscularly) 24 h before AI. Compared with the no treatment (Control) cows, ECP treatment increased (P < 0.01) pregnancy rates of cows induced to ovulate smaller dominant follicles (<12.2 mm). In conclusion, GnRH-induced ovulation of small dominant follicles was associated with reduced serum estradiol, fertilization rate (donor cows), and pregnancy establishment (recipient cows). Furthermore, ECP supplementation during the preovulatory period increased pregnancy rates in cows induced to ovulate smaller dominant follicles.

Factors affecting preovulatory follicle diameter and ovulation rate after gonadotropin-releasing hormone in postpartum beef cows. Part I: Cycling cows

Pregnancy failure in livestock can result from failure to fertilize the oocyte or embryonic loss during gestation. The focus of this review is on cattle and factors affecting and mechanisms related to uterine insufficiency for pregnancy. A variety of factors contribute to embryonic loss and it may be exacerbated in certain animals, such as high-producing lactating dairy cows, and in some cattle in which estrous synchronization and timed AI was performed, due to reduced concentrations of reproductive steroids. Recent research in beef cattle induced to ovulate immature follicles and in lactating dairy cows indicates that deficient uterine function is a major factor responsible for infertility in these animals. Failure to provide adequate concentrations of estradiol before ovulation results in prolonged effects on expression and localization of uterine genes and proteins that participate in regulating uterine functions during early gestation. Furthermore, progesterone concentrations during early gestation affect embryonic growth, interferon-tau production, and uterine function. Therefore, an inadequate uterine environment induced by insufficient steroid concentrations before and after ovulation could cause early embryonic death either by failing to provide an adequate uterine environment for recognition of embryo signaling, adhesion, and implantation or by failing to support appropriate embryonic growth, which could lead to decreased conceptus size and failed maternal recognition of pregnancy.

Effects of Fertility on Gene Expression and Function of the Bovine Endometrium

Cows induced to ovulate small dominant follicles were reported to have reduced pregnancy rates compared with cows that ovulated large follicles. The reason for the presence of small dominant follicles at the time of GnRH-induced ovulation in timed AI protocols is unknown. The objectives of this experiment were to examine the role of day of the estrous cycle at initiation of treatment on ovulation after the first GnRH injection (GnRH1) and associated effects on growth rate and final size of the ovulatory follicle at the second GnRH injection (GnRH2), serum concentrations of estradiol at GnRH2, and subsequent luteal concentrations of progesterone in suckled beef cows. Estrous cycles of cows were manipulated to be at 1 of 5 specific days of the cycle (d 2, 5, 9, 13, and 18, d 0 = estrus; n = 12 per treatment group) at the beginning of the CO-Synch protocol (GnRH1 on d -9, PGF(2alpha) on d -2, and GnRH2 on d 0). Day of the estrous cycle at GnRH1 did not affect the size of the preovulatory follicle or the proportion of cows ovulating at GnRH2 (P = 0.65 and 0.21, respectively). When all cows were included in the analysis, cows that ovulated after GnRH1 had similar follicle size at GnRH2 compared with cows that did not ovulate after GnRH1 (11.4 and 10.4 mm, respectively; P = 0.23). When only cows that could ovulate after GnRH1 (excluding cows treated on d 2) were included in the analysis, cows that ovulated to GnRH1 had a larger follicle at GnRH2 than cows that did not ovulate after GnRH1 (11.4 and 9.5 mm, respectively; P = 0.04). Follicle growth from d -5 to 0 was similar between cows that ovulated after GnRH1 and cows that did not (1.01 vs. 0.89 mm/d, respectively; P = 0.75). There was a tendency for faster follicle growth rate in cows that ovulated a large follicle (>11 mm) compared with cows that ovulated a small follicle (< or = 11 mm; 1.01 vs. 0.86 mm/d, respectively; P = 0.07). Serum concentrations of estradiol at GnRH2 and progesterone after ovulation were reduced in cows that ovulated small follicles compared with cows that ovulated large follicles (P = 0.006 and 0.005, respectively). In summary, day of the estrous cycle at initiation of synchronization did not affect ovulatory follicle size, but follicle growth rates affected the size of the follicle at GnRH2. Cows that ovulated a small follicle had reduced serum concentrations of estradiol at GnRH2 and progesterone after ovulation.

Pregnancy establishment and maintenance in cattle.

Infertility and subfertility are important and pervasive reproductive problems in both domestic animals and humans. The majority of embryonic loss occurs during the first three weeks of pregnancy in cattle and women due, in part, to inadequate endometrial receptivity for support of embryo implantation. To identify heifers of contrasting fertility, serial rounds of artificial insemination (AI) were conducted in 201 synchronized crossbred beef heifers. The heifers were then fertility classified based on number of pregnancies detected on day 35 in four AI opportunities. Heifers, classified as having high fertility, subfertility or infertility, were selected for further study. The fertility-classified heifers were superovulated and flushed, and the recovered embryos were graded and then transferred to synchronized recipients. Quantity of embryos recovered per flush, embryo quality, and subsequent recipient pregnancy rates did not differ by fertility classification. Two in vivo-produced bovine embryos (stage 4 or 5, grade 1 or 2) were then transferred into each heifer on day 7 post-estrus. Pregnancy rates were greater in high fertility than lower fertility heifers when heifers were used as embryo recipients. The reproductive tracts of the classified heifers were obtained on day 14 of the estrous cycle. No obvious morphological differences in reproductive tract structures and histology of the uterus were observed in the heifers. Microarray analysis revealed differences in the endometrial transcriptome based on fertility classification. A genome-wide association study, based on SNP genotyping, detected 7 moderate associations with fertility across 6 different chromosomes. Collectively, these studies support the idea that innate differences in uterine function underlie fertility and early pregnancy loss in ruminants. Cattle with defined early pregnancy success or loss is useful to elucidate the complex biological and genetic mechanisms governing endometrial receptivity and uterine competency for pregnancy.

Effects of flunixin meglumine on pregnancy establishment in beef cattle.

A single ovulation, reciprocal embryo transfer study was used to investigate effects of oocyte competence and maternal environment on pregnancy establishment and maintenance in beef cows. Estrous cycles were synchronized in suckled beef cows and embryo donors were inseminated on d 0 (n = 810). Cows were classified on d 0 as having a small (<12.5 mm) or large (≥12.5 mm) ovulatory follicle and randomly chosen as donors or recipients to remove confounding effects of ovulatory follicle size on fertility. Embryos (n = 393) or oocytes (n = 44) were recovered on d 7, and all viable embryos were transferred into recipients (n = 354). All statistical analyses were conducted using the GLM procedure of SAS. Path analysis (with significance set at P < 0.10) was used to examine potential cause-effect relationships among the measured variables. Greater donor cow BW, circulating estradiol concentration at insemination, postpartum interval, and ovulatory follicle size directly increased (P < 0.10) fertilization success. Greater donor cow age was the only factor that directly decreased (P < 0.10) fertilization success. Viability of d-7 embryos was directly inhibited (P < 0.10) by rapid follicular growth rate from d -2 to 0 and heavier BW. Direct beneficial effects to embryo viability were increased serum progesterone concentration on d -2 and ovulatory follicle size. Pregnancy maintenance from d 7 to 27 was enhanced (P < 0.10) by increased serum estradiol concentration on d 0 and progesterone concentration on d 7 in the recipient cow. Increased follicular diameter in the recipient cow on d 0 was detrimental to pregnancy maintenance from d 7 to 27. This manuscript defines the complex interplay and relative contributions of endocrine and physical factors both prior and subsequent to fertilization that influence both oocyte competence and maternal environment and their roles in establishment and maintenance of pregnancy.

Increased Conception Rates in Beef Cattle Inseminated with Nanopurified Bull Semen

The objective of this research was to determine effects of a single injection of the PG synthesis inhibitor flunixin meglumine (FM; 1.1 mg/kg of BW, intramuscularly) approximately 13 d (range 10 to 15 d) after AI on pregnancy establishment. Three experiments were conducted using estrus-synchronized heifers and cows. Technicians and AI sires were equally represented across treatments within locations and experiments. Bulls were introduced on the day of FM treatment. Pregnancy to AI was diagnosed 28 to 50 d after AI using ultrasonography. In Exp. 1, beef heifers (n = 1,221) were divided within 5 locations to receive FM or no further treatment (control). At insemination, heifers were divided into 2 similar pastures or pens, and approximately 13 d later, 1 group of heifers within each location was processed through an animal handling facility to administer FM treatment. There was no location x treatment interaction (P = 0.62) on AI pregnancy rates, so data were pooled. Pregnancy rates to AI were reduced (P = 0.02) among heifers receiving the FM treatment procedure (66%) compared with control heifers (72%). In Exp. 2, suckled beef cows (n = 719) were assigned within 2 locations to receive FM or no further treatment (control) approximately 13 d after AI. At insemination, control and FM cows were divided into separate pastures, and only FM cows were handled after AI for the FM treatment procedure. There was no location x treatment interaction (P = 0.75), so data were pooled. Pregnancy rates to AI did not differ (P = 0.80) between FM (57%) and control cows (59%). In Exp 3, beef heifers (n = 247) and suckled beef cows (n = 335) from 1 location received no injection (control) or injection of FM approximately 13 d after AI when all cows and heifers were processed through a working facility. Pregnancy rates to AI were not different (P = 0.37) between FM (45%) and control (42%) cows or between FM (56%) and control (55%) heifers. We conclude FM administration at 1.1 mg/kg of BW approximately 13 d after AI did not improve pregnancy establishment in beef cows and heifers and that the effects of handling heifers at this time may decrease pregnancy establishment.

Triennial Reproduction Symposium: influence of follicular characteristics at ovulation on early embryonic survival.

ABSTRACT Aberrant sperm phenotypes coincide with the expression of unique sperm surface determinants that can be probed by objective, biomarker-based semen analysis and targeted as ligands for semen purification. This study evaluated a nanoparticle-based magnetic purification method that removes defective spermatozoa (∼30% of sample) from bull semen and improves sperm sample viability and fertilizing ability in vitro and in vivo. Two types of nanoparticles were developed: a particle coated with antibody against ubiquitin, which is present on the surface of defective spermatozoa, and a particle coated with the lectin peanut agglutinin, which binds to glycans exposed by acrosomal damage. In a 2 yr artificial insemination field trial with 798 cows, a conception rate of 64.5% ± 3.7% was achieved with a 10 × 106 sperm dose of peanut agglutinin-nanopurified spermatozoa, comparable to a control nonpurified full dose of 20 × 106 spermatozoa per dose (63.3% ± 3.2%) and significantly higher than a 10 × 106 sperm dose of nonpurified control semen (53.7% ± 3.2%; P < 0.05). A total of 466 healthy calves were delivered, and no negative side effects were observed in the inseminated animals or offspring. Because the method is inexpensive and can be fully integrated in current protocols for semen cryopreservation, it is feasible for use in the artificial insemination industry to improve fertility with reduced sperm dosage inseminations. Spermatology will benefit from nanopurification methodology by gaining new tools for the identification of candidate biomarkers of sperm quality such as binder of sperm protein 5 (BSP5), described in the present study.