Timed Embryo Transfer Research Articles

Efficacy of in vitro embryo transfer in lactating dairy cows using fresh or vitrified embryos produced in a novel embryo culture medium

Objectives were to determine whether pregnancy success could be improved in lactating cows with timed embryo transfer when embryos were produced in vitro using a medium designed to enhance embryo development and survival after cryopreservation. In experiment 1, embryos (n=569 to 922) were cultured in either modified synthetic oviduct fluid or a serum-free medium, Block-Bonilla-Hansen-7 (BBH7). Development to the blastocyst stage was recorded at d 7, and selected blastocysts (n=79 to 114) were vitrified using open pulled straws. Culture of embryos in BBH7 increased development to the blastocyst stage (41.9±2.0 vs. 14.7±2.0%) and advanced blastocyst stages (expanded, hatching, hatched; 31.1±1.3 vs. 6.4±1.3%) at d 7 and resulted in higher hatching rates at 24h postwarming compared with embryos cultured in modified synthetic oviduct fluid (59.0±0.5 vs. 26.7±0.5%). In experiment 2, embryos were produced using X-sorted semen and cultured in BBH7. At d 7 after insemination, embryos were transferred fresh or following vitrification. Lactating Holstein cows were either subjected to timed artificial insemination (TAI) on the day of presumptive ovulation or used as embryo recipients 7 d later. Embryo recipients received an embryo if a corpus luteum was present. The percentage of cows pregnant at d 32, 46, and 76 of gestation was higher among cows that received fresh embryos compared with TAI cows or cows that received vitrified embryos. At d 76, for example, the proportion and percentage pregnant was 47/150 (31.3%) for cows subjected to TAI, 48/95 (50.5%) for cows receiving fresh embryos, and 39/141 (27.7%) for cows receiving a vitrified embryo. No difference was observed in the percentage of cows pregnant among TAI cows and those that received vitrified embryos. There was a service or transfer number × treatment interaction because differences in pregnancy rate between embryo transfer recipients and cows bred by TAI were greater for cows with more than 3 services or transfers. Pregnancy success in lactating cows can be improved by transferring fresh embryos produced in BBH7 compared with TAI. Moreover, no decline in fertility was observed when cryopreserved embryos were transferred compared with TAI. Embryo transfer is particularly efficacious for infertile cows that have previously experienced several failed breeding attempts.

The applications of timed artificial insemination and timed embryo transfer in reproductive management of dairy cattle

Fertility of dairy cattle is generally decreasing. Overall estrus detection efficiency in lactating dairy cattle is low, as expression of estrus is often compromised. Consequently, undetected estrus, low AI-submission rates, and long inter-breeding intervals are the main contributors to poor reproductive efficiency. Although failure to become pregnant is the most common reason for culling dairy cattle, pregnancy rates could be improved by increasing the AI-submission rate through increased estrus detection efficiency, timed insemination (Timed-AI), or timed embryo transfer (Timed-ET). In these protocols, ovarian follicular development and luteolysis are controlled, culminating in synchronous ovulation in most cows. There are several modifications to improve pregnancy rates in Timed-AI protocols, e.g. presynchronization with 2 doses of PGF2a 14 d apart, and starting the Ovsynch protocol 12 d after the 2nd PGF2a. Timed-AI programs ensure a timely first breeding; this should be followed with early pregnancy diagnosis to identify nonpregnant cows, and prompt re-insemination. However, in a recent field study involving 23 dairy herds, the mean interval between 1st and 2nd breeding was 42 d; only 28% of the cows were rebred within 24 d after 1st breeding. Fortunately, there are several resynchronization protocols to reduce inter-breeding intervals. Anestrus cows, or those with cystic ovarian follicles, have a good chance of conceiving when subjected to synchronization of ovulation and Timed-AI. Furthermore, Timed-ET is a practical and viable option for synchronizing and selecting recipient cattle for embryo transfers. In summary, systematic and judicious use of Timed-AI and Timed-ET protocols can greatly enhance reproductive performance, and improve herd productivity.

144 USE OF A NOVEL BOVINE EMBRYO CULTURE MEDIUM TO IMPROVE BLASTOCYST DEVELOPMENT AND SURVIVAL FOLLOWING VITRIFICATION

The objective of the present study was to determine whether culture of bovine embryos in a proprietary serum-free culture medium, Block-Bonilla-Hansen-7 (BBH-7), could improve development to the blastocyst stage and enhance survival following vitrification. For Exp. 1, embryos were produced in vitro and cultured in BBH-7 or modified synthetic oviductal fluid (mSOF; as in zygote 10:341 except with 10 μL mL-1 of nonessential amino acids, 20 μL mL-1 of essential amino acids, and 1 mg mL-1 of polyvinyl alcohol instead of albumin) in 5% (v/v) oxygen. Grade 1 expanded blastocysts were harvested at Day 7 post-insemination and vitrified using the open-pulled straw method (Vagta et al. 1998 Mol. Reprod. Dev. 51, 53-58). Vitrified embryos were thawed and cultured in vitro in either mSOF or BBH-7 supplemented with 10% fetal bovine serum and 50 μM dithiolthreitol. Re-expansion and hatching rates were recorded at 24, 48, and 72 h post-thaw. There was no effect of culture medium on cleavage rate. The proportion of oocytes that developed to the blastocyst and advanced blastocyst stages (expanded, hatching, and hatched) at Day 7 was higher (P < 0.001) for embryos cultured in BBH-7 than for embryos cultured in mSOF (41.9 ± 2.0 v. 14.7 ± 2.0% and 31.1 ± 1.3 v. 6.4 ± 1.3%, respectively). There was no effect of culture medium on re-expansion rates at 24, 48, and 72 h post-thaw or on hatching rates at 48 or 72 h. However, the proportion of embryos that were hatching or had hatched by 24 h post-thaw was higher (P < 0.001) for BBH-7 than for mSOF (59.0 ± 0.5 v. 26.7 ± 0.5%). For Exp. 2, late lactation and/or repeat breeder, lactating Holstein cows were synchronized for timed embryo transfer using the OvSynch-56 protocol. Embryos were produced in vitro and cultured in BBH-7 in 5% (v/v) oxygen. Vitrified embryos were produced as for Exp. 1. Fresh embryos were grade 1 expanded blastocysts harvested at Day 7 after insemination. A single embryo was transferred at Day 7 after putative ovulation to all cows with a corpus luteum confirmed by ultrasonography. Pregnancy was diagnosed at Day 28-30 of gestation by ultrasonography. There was no difference in the proportion of recipients that became pregnant after receiving either a fresh (7/18 = 39%) or vitrified (10/27 = 37%) embryo cultured in BBH-7. The results of the present study indicate that BBH-7 can be used to increase the proportion of oocytes that develop to the blastocyst stage. Moreover, the results demonstrate that vitrified embryos produced after culture in BBH-7 can achieve pregnancy rates similar to those obtained using fresh embryos. Support: USDA 2006-55203-17390 and Southeast Milk Checkoff Program.

145 FACTORS AFFECTING PREGNANCY RATE FOLLOWING TIMED EMBRYO TRANSFER PROGRAM IN CATTLE UNDER FIELD CONDITIONS

Hormonal estrus synchronization followed by heat detection of recipient animals is a widely used technique in embryo transfer (ET) programs around the world. However, the successful outcomes of ET rely heavily on appropriate heat detection of recipient animals. Additionally, limited availability of personnel and time to detect heat makes ET impractical at times. Therefore, pregnancy rates (PR) following a timed embryo transfer (TET) program were evaluated taking into account different variables of recipient cows, donor embryos, and season of transfer under field conditions in Argentina. Recipient cows were synchronized utilizing a progesterone (P4) intravaginal device (DIB, Syntex, Argentina) for 7 days. On Day 0 (day of DIB insertion) 2 mg of estradiol benzoate (EB) i.m. was administered and immediately after DIB removal on Day 7, 500 μg of D(+) Cloprostenol i.m. (PG, Ciclase, Syntex), 400 IU of eCG i.m. (Novormon, Syntex) plus 1 mg of cypionate estradiol i.m. (ECP; König, Agentina) were administered to all recipient cows. Fresh (n = 1746) and frozen (n = 2047) beef bovine embryos were nonsurgically transferred into recipient cows on Day 9 after PG. The effects of body condition score (1–9, BCS I: ≤5, n = 804 and BCS II: ≥6, n = 2989), corpus luteum (CL) score (CL1: prominent papilla, n = 932, CL2: small papilla, n = 2028, and CL3: asymmetric, n = 833), location of embryo placement in uterine horn (upper, n = 3566, and lower, n = 227), and type of recipient such as heifer (n = 487), lactating (n = 1010), or dry cows (n = 2296) on PR were evaluated. Additionally, stage of embryo development (4–7), quality (1–3), and season of transfer (spring, summer, fall, and winter) were included in the analysis. Pregnancy rate was confirmed by rectal palpation 60 days after ET. Data were first screened by chi square and then analyzed by Proc Logistic of SAS (SAS Institute Inc., Cary, NC). Overall PR was 54.7%. body condition score, CL, type of recipients, stage of embryo development, and year were not significantly (P > 0.05) associated with PR. As expected, cows that received direct fresh embryos had 1.49 times greater odds of conceiving (odds ratio, OR = 1.49, 95% CI: 1.22 to 1.82) than those receiving frozen embryos. Additionally, cows receiving embryos graded as quality 1 had greater probability to conceive (OR = 1.62, 95% CI: 1.34 to 1.95, or OR = 1.64, 95% CI: 1.25 to 2.21) compared to quality 2 or 3 respectively. Moreover, embryos placed into the upper portion of uterine horn were more likely (OR = 1.48) to conceive than those placed in the lower uterine portion. Regardless of embryos and recipient cow variables analyzed, there was a greater probability for cows to conceive during winter (OR = 1.41, 95% CI: 1.13 to 1.95) as compared with summer. In conclusion, synchronized recipient animals for a TET program resulted in acceptable PR for both fresh and frozen embryos. Results suggested that the quality and location of embryos (upper portion) in uterine horn were the most important variables associated with PR in recipient cows. Therefore, TET is a viable and practical option allowing a successful outcome for many beef operations where availability of personnel is limited.

The effect of in vitro treatment of bovine embryos with IGF-1 on subsequent development in utero to Day 14 of gestation

Culture of bovine embryos with insulin-like growth factor-1 (IGF-1) can improve development to the blastocyst stage and embryo survival following transfer to heat-stressed, lactating dairy cows. Two experiments were conducted to determine whether IGF-1 could improve embryo survival and development at Day 14 after ovulation. In Experiment 1, non-lactating Holstein cows ( n = 58) were selected as recipients following synchronization for timed-embryo transfer. Embryos were produced in vitro and cultured with or without 100 ng/mL IGF-1. At Day 7 after expected ovulation (Day 0), groups of 7–12 embryos were randomly transferred to each recipient. Embryos were recovered at Day 14. Embryo length and the presence or absence of an embryonic disc was recorded. Recovered embryos were cultured individually for 24 h to determine interferon-τ (IFN-τ) secretion. There was no effect of IGF-1 on embryo recovery rate, embryo length or IFN-τ secretion. In Experiment 2, non-lactating ( n = 56) and lactating ( n = 35) Holstein cows were selected as recipients following synchronization for timed-embryo transfer. Embryos were produced as described in Experiment 1. At Day 7 after expected ovulation (Day 0), a single embryo was randomly transferred to each recipient. Embryos were recovered at Day 14. Embryo length and IFN-τ secretion were determined as in Experiment 1. Recovery rate at Day 14 tended ( P = 0.1) to be higher for recipients that received IGF-1 treated embryos compared to control embryos (43.2% versus 26.1%, respectively). There was no effect of IGF-1 on embryo length or IFN-τ secretion. In conclusion, results suggest that exposure to IGF-1 through Days 7–8 of development does not enhance capacity of embryos to prevent luteolysis. Results of the single embryo-transfer experiment suggested that IGF-1 treatment might affect embryo survival post-transfer as early as Day 14 after ovulation. Further experimentation is warranted to verify this finding.

Interaction between season and culture with insulin-like growth factor-1 on survival of in vitro produced embryos following transfer to lactating dairy cows

Culture of bovine embryos in the presence of insulin-like growth factor-1 (IGF-1) can increase pregnancy rates following transfer to heat-stressed, lactating dairy cows. The objective of the present experiment was to determine whether the effect of IGF-1 on post-transfer embryo survival was a general effect or one specific to heat stress. Lactating recipients ( n = 311) were synchronized for timed-embryo transfer at four locations. Embryos were produced in vitro and cultured with or without 100 ng/mL IGF-1. At Day 7 after anticipated ovulation (Day 0), a single embryo was randomly transferred to each recipient. Pregnancy was diagnosed at Day 21 by elevated plasma progesterone concentrations, at Days 27–32 by ultrasonography, and at Days 41–49 by transrectal palpation. Transfers were categorized into two seasons, hot or cool (based on the month of transfer). There was a tendency ( P < 0.09) for an interaction between embryo treatment and season for pregnancy rate at Day 21; this interaction was significant at Days 30 and 45 ( P < 0.02). Recipients receiving IGF-1 treated embryos had higher pregnancy rates in the hot season but not in the cool season. There was a similar interaction between embryo treatment and season for overall calving rate ( P < 0.05). There was also an interaction between season and treatment affecting pregnancy loss between Days 21 and 30; recipients that received IGF-1 treated embryos had less pregnancy loss during this time period in the hot season but not in the cool season. The overall proportion of male calves born was 77.5%. In conclusion, treatment of embryos with IGF-1 improved pregnancy and calving rates following the transfer of in vitro produced embryos into lactating recipients, but only under heat-stress conditions.

Control of ovarian follicular and corpus luteum development for the synchronization of ovulation in cattle

The objective of this review is to integrate strategies to optimize an ovulatory control program which then serves as a platform to improve the reproductive performance of lactating dairy cows. Programmed management of follicle growth, regression of the CL and induction of ovulation led to development of the Ovsynch program. Pre-synchronization of estrous cycles followed 12 to 14 days later with the Ovsynch program increased pregnancy rates to timed inseminations. Initiation of the Ovsynch program on day 3 of the estrous cycle reduced ovulation to GnRH and resulted in a smaller proportion of excellent and good quality embryos following timed insemination. The pregnancy rate to a timed insemination of Ovsynch was greater when cows ovulated to the first injection of GnRH. The Presynch-Ovsynch program provided a platform to identify factors regulating reproductive performance; such as, parity, body condition score and anovulation. Treatment with hCG at day 5 after insemination increased pregnancy rate in lactating dairy cows. Injection of bovine somatotropin at insemination increased pregnancy rate, conceptus length and interferon-tau content in uterine luminal flushings and altered endometrial gene expression at day 17 of pregnancy. During heat stress, timed embryo transfer increased pregnancy rate and using embryos cultured with IGF-I and transferred fresh resulted in a greater pregnancy rate. Induction of ovulation with estradiol cypionate, as a component of a timed insemination program, increased fertility. Manipulation of the estrous cycle to improve follicle/oocyte competence and management of the post-ovulatory dialogue between embryonic and uterine tissues should enhance embryo development and survival.

Ovsynch Plus CIDR Protocol for Timed Embryo Transfer in Suckled Postpartum Japanese Black Beef Cows

We compared synchronization and pregnancy rates, and the increase in blood progesterone concentrations during luteal development, between (1) Ovsynch plus an intravaginal controlled internal drug release (CIDR) device protocol followed by timed embryo transfer (timed ET), and (2) a conventional estrus synchronization method using PGF(2 alpha) and ET in suckled postpartum Japanese Black beef cows. Cows in the PGF group (n=18) received a PGF(2 alpha) analogue when a CL was first palpated per rectum at 10-d intervals after 1 to 2 month postpartum. Cows (n=11), which showed estrus (Day 0) within 5 d of the PGF(2 alpha), and had a CL on Day 7, received ET. Cows in the Ovsynch+CIDR group (n=19) underwent the Ovsynch protocol plus a CIDR for 7 d (GnRH analogue and CIDR on Day-9, PGF(2alpha) analogue with CIDR removal on Day-2, and GnRH analogue on Day 0), with ET on Day 7. The ovulation synchronization (100%) and embryo transfer (100%) rates in the Ovsynch+CIDR group were greater (P<0.01) than the estrus synchronization (66.7%) and the embryo transfer (61.1%) rates in the PGF group. The postpartum interval at ET in the Ovsynch+CIDR group (62.5 +/- 2.5 d) was shorter (P<0.01) than in the PGF group (74.9 +/- 3.9 d). The pregnancy rate in the Ovsynch+CIDR group (57.9%) did not differ significantly from that in the PGF group (50.0%). Plasma progesterone concentrations were not significantly different in the two groups on Days 0, 1, 2, 5, 7, 14 and 21. In summary, higher synchronization and transfer rates, and shorter postpartum interval to ET, can be achieved with timed ET following the Ovsynch plus CIDR protocol than after estrus with the single PGF(2 alpha) treatment followed by ET in suckled postpartum recipient beef cows. Pregnancy rates were similar. Also, the increase in blood progesterone concentrations during luteal development following ovulation synchronized by the Ovsynch plus CIDR protocol was similar to that after estrus induced by the PGF(2 alpha) treatment.

208 EFFECT OF GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR ON BLASTOCYST DEVELOPMENT AND POST-TRANSFER SURVIVAL OF IN VITRO-PRODUCED BOVINE EMBRYOS

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that has been implicated in preimplantation embryo development. Granulocyte-macrophage-CSF improves the proportion of bovine embryos that become blastocysts in vitro (Moraes and Hansen 1997 Biol. Reprod. 57, 1060–1065) and increases blastocyst cell numbers in mice (Robertson et al. 2001 Biol. Reprod. 64, 1206–1215). The long-term goal of the present research was to evaluate the effects of GM-CSF on post-transfer survival of bovine embryos. The experiments used recombinant ovine GM-CSF produced in transfected Chinese hamster ovary (CHO) cells or an equivalent volume of cytokine-free CHO cell supernatant (control). The objective of the first study was to evaluate the effects of GM-CSF on post-transfer survival. Embryos were cultured with 10 ng mL–1 of either GM-CSF or cytokine-free CHO cell supernatant added to culture medium at Day 1 after insemination. Embryos were transferred at Day 7 to lactating dairy cows according to a timed embryo transfer protocol. Pregnancy was evaluated at approximately Day 45 of gestation. There was no significant difference in the proportion of embryos becoming blastocysts at Day 7 after insemination (34.8 v. 37.5% for the control and GM-CSF; SEM = 2.4%). There was also no difference in pregnancy rates between cows receiving control embryos (6/24; 25%) and cows receiving embryos treated with GM-CSF (8/35; 23%). A second study determined the effects of various concentrations of GM-CSF on the development of in vitro-produced embryos to the blastocyst stage. Embryos were cultured in 5% (v/v) oxygen (low oxygen) or atmospheric oxygen (21%, w/v; high oxygen) in the presence of 0, 1, 10, or 100 ng mL–1 of GM-CSF or an equivalent volume of cytokine-free CHO cell supernatant (control). The GM-CSF was added on either Day 1 or Day 5 after insemination. Cleavage rate was accessed on Day 3 after insemination. Stage of development was recorded at Day 7 and Day 8 after insemination. There was no effect of GM-CSF on cleavage rate. Addition of GM-CSF at Day 5 to embryos cultured in low or high oxygen increased the percentage of oocytes that became blastocysts at Day 7 (P &lt; 0.01) and Day 8 (P &lt; 0.01), but addition at Day 1 did not have a significant effect on blastocyst development. The greatest effects of GM-CSF occurred at a concentration of 10 ng mL–1. At this concentration, least squares means for the percentage of oocytes that became blastocysts at Day 7 were 13.9 v. 21.6% (control v. GM-CSF) when GM-CSF was added at Day 5, and 19.5 v. 21.5% when GM-CSF was added at Day 1. The percentage of blastocysts at Day 8 was 20.9 v. 28.7% when GM-CSF was added at Day 5, and 26.7 v. 27.5% when GM-CSF was added at Day 1. In conclusion, GM-CSF can affect the competence of embryos to develop to the blastocyst stage, but at the concentrations and times given, there was no evidence that GM-CSF enhanced embryo survival after transfer.

A CIDR-Based Timed Embryo Transfer Protocol Increases the Pregnancy Rate of Lactating Repeat Breeder Dairy Cows

This study evaluated the pregnancy rates following either a controlled internal drug release (CIDR)-based timed artificial insemination (TAI) or an embryo transfer (TET) protocol compared with that following a single PGF(2alpha) injection and AI after estrus (AIE) in lactating repeat breeder dairy cows. Fifty-three lactating dairy cows diagnosed as repeat breeders were used in this study and were randomly assigned to the following three treatments. (1) Cows, at random stages of the estrous cycle, received a CIDR device and 2 mg estradiol benzoate (EB; Day 0), a 25 mg PGF(2) (alpha) injection at the time of CIDR removal on Day 7 and a 1 mg EB injection on Day 8. The cows then received TAI 30 h (Day 9) after the second EB injection using dairy semen (TAI group, n=13). (2) Cows, at random stages of the estrous cycle, received the same hormonal treatments as in the TAI group. The cows then received TET on Day 16 using frozen-thawed blastocysts or morula embryos collected from Korean native cattle donors (TET group, n=13). (3) Cows, at the luteal phase, received a 25 mg injection of PGF(2alpha) and AIE using dairy semen (control group, n=27). The ovaries of the cows in the TET group were examined by transrectal ultrasonography to determine ovulation of the preovulatory follicles, and blood samples were collected for serum progesterone (P4) analysis. The pregnancy rate was significantly higher in the TET group (53.8%) than in the control (18.5%) or TAI (7.7%) groups (P<0.05). The ultrasonographic observations demonstrated that all the cows in the TET group ovulated the preovulatory follicles and concomitantly formed new corpora lutea. Accordingly, the mean serum P4 concentration remained constant between Day 0 and Day 7 of the luteal phase, decreased dramatically on Day 8 (P<0.01) and subsequently increased by Day 16 (P<0.01). These data suggest that the CIDR-based TET protocol can be used to effectively increase the pregnancy rate in lactating repeat breeder dairy cows.

18 THE EFFECTS OF CIDR AND eCG TREATMENTS IN A GnRH-BASED PROTOCOL FOR TIMED AI OR EMBRYO TRANSFER ON PREGNANCY RATES IN LACTATING BEEF COWS

Two experiments were conducted to determine the effects of the addition of a progestin, equine chorionic gonadotropin (eCG), or both, in a GnRH-based protocol for timed AI (TAI) or timed embryo transfer (TET). In both experiments, Angus, Gelbvieh, and Simmental cross-bred cows were randomized by breed and postpartum interval [50 � 10 days (mean � SD); range, 27 to 89] into 4 groups in a 2 � 2 factorial design. All injections were given IM. In Experiment 1, 288 cows (89.6% cycling) were given 25 mg dinoprost (PGF; Lutalyse�; Pfizer Animal Health, Montreal, Quebec, Canada) on Day –11; on Day 0, they were given 100 �g GnRH (Cystorelin�; Merial Canada, Pointe-Claire, Quebec, Canada), with or without concomitant insertion of a CIDR (1.9 g progesterone; Pfizer Animal Health, Montreal, Quebec, Canada). On Day 7, CIDR inserts were removed and cows were given PGF, with or without concomitant injection of 400 IU of eCG (Pregnecol�; Bioniche Animal Health, Belleville, Ontario, Canada). On Day 9 (54-56 h after PGF), TAI was done, with concomitant injection of 100 �g GnRH. Ultrasonographic examination of 147 cows on Day 7 revealed that 62.4% had ovulated in response to the first GnRH. Pregnancy rates (ultrasonographic examination) on Day 38 did not differ between cows with or without a CIDR (52.9 and 51.4%, rspectively; P ≥ 0.64), with or without eCG treatment (53.5 and 50.7%, respectively; P ≥ 0.28), in cycling vs. anestrous cows (51.6 vs 56.7%, respectively; P ≥ 0.76), and in cows that had ovulated (58.1%) or did not ovulate (50.0%) after the first GnRH treatment (P ≥ 0.58). In Experiment 2, 151 cows were given 500 �g cloprostenol (PGF; Estrumate�; Schering–Plough Animal Health, Pointe-Claire, Quebec, Canada) on Day –12, 100 �g GnRH on Day 0, with or without concomitant insertion of a CIDR. On Day 3, half of the cows were given 400 IU eCG. On Day 7, CIDRs were removed and cows were given PGF; on Day 9 (54–56 h after PGF), all cows were given 100 �g GnRH. On Day 15, ultrasonography was done to select suitable recipients for transfer of frozen–thawed embryos on Day 16 (part of another experiment, balanced across synchronization groups). Recipient selection rates did not differ whether cows received or did not receive a CIDR (93.4% vs 85.5%, respectively; P ≥ 0.27) or eCG (91.0 vs 87.8%, respectively; P ≥ 0.67). In addition, pregnancy rates on Day 43 did not differ whether cows received or did not receive a CIDR (32.3 vs 32.4%, respectively; P ≥ 0.52) or eCG (35.2 and 29.2%, respectively; P ≥ 0.21). In summary, the addition of a CIDR or eCG to a GnRH-based synchronization protocol initiated after PGF presynchronization in lactating beef cattle yielded no improvement in pregnancy rates following TAI, or recipient selection and pregnancy rates following TET.

Effect of transfer of one or two in vitro-produced embryos and post-transfer administration of gonadotropin releasing hormone on pregnancy rates of heat-stressed dairy cattle

Pregnancy rates following transfer of an in vitro-produced (IVP) embryo are often lower than those obtained following transfer of an embryo produced by superovulation. The purpose of the current pair of experiments was to examine two strategies for increasing pregnancy rates in heat stressed, dairy recipients receiving an IVP embryo. One method was to transfer two embryos into the uterine horn ipsilateral to the CL, whereas the other method involved injection of GnRH at Day 11 after the anticipated day of ovulation. In Experiment 1, 32 virgin crossbred heifers and 26 lactating crossbred cows were prepared for timed embryo transfer by being subjected to a timed ovulation protocol. Those having a palpable CL were randomly selected to receive one ( n = 31 recipients) or two ( n = 27 recipients) embryos on Day 7 after anticipated ovulation. At Day 64 of gestation, the pregnancy rate tended to be higher ( P = 0.07) for cows than for heifers. Heifers that received one embryo tended to have a higher pregnancy rate than those that received two embryos (41% versus 20%, respectively) while there was no difference in pregnancy rate for cows that received one or two embryos (57% versus 50%, respectively). Pregnancy loss between Day 64 and 127 only occurred for cows that received two embryos (pregnancy rate at Day 127 = 17%). Between Day 127 and term, one animal (a cow with a single embryo) lost its pregnancy. There was no difference in pregnancy rates at Day 127 or calving rates between cows and heifers, but females that received two embryos had lower Day-127 pregnancy rates and calving rates than females that received one embryo ( P < 0.03). Of the females receiving two embryos that calved, 2 of 5 gave birth to twins. For Experiment 2, 87 multiparous, late lactation, nonpregnant Holstein cows were synchronized for timed embryo transfer as in Experiment 1. Cows received a single embryo in the uterine horn ipsilateral to the ovary containing the CL and received either 100 μg GnRH or vehicle at Day 11 after anticipated ovulation (i.e. 4 days after embryo transfer). There was no difference in pregnancy rate for cows that received the GnRH or vehicle treatment (18% versus 17%, respectively). In conclusion, neither unilateral transfer of two embryos nor administration of GnRH at Day 11 after anticipated ovulation improved pregnancy rates of dairy cattle exposed to heat stress.

189 PREGNANCY RATES IN HEAT-STRESSED DAIRY CATTLE RECEIVING ONE OR TWO IN VITRO-PRODUCED EMBRYOS IN A TIMED EMBRYO TRANSFER PROGRAM

The objective was to determine whether transfer of two embryos would increase pregnancy rates in heat-stressed dairy recipients receiving an in vitro-produced embryo transferred into the uterine horn ipsilateral to the corpus luteum (CL). Such a treatment would increase the likelihood that the cow receives at least one embryo competent for sustained development. In addition, transfer of two embryos into the ipsilateral uterine horn is likely to increase the amounts of interferon-tau and other embryonic-signaling molecules in the uterus. A total of 32 virgin crossbreed heifers and 26 lactating crossbreed cows were used as timed embryo transfer recipients after being subjected to an ovulation synchronization protocol as follows: GnRH (100 �g) and insertion of previously used progesterone-containing CIDR on Day -10, prostaglandin F2� and CIDR removal on Day -3, and GnRH (100 �g) on Day 0 (day of anticipated ovulation). All recipients had a palpable CL on Day 6 and were randomly selected to receive one (n = 31 recipients) or two (n = 27) embryos on Day 7. At Day 64, the pregnancy rate tended to be higher (P = 0.07) for cows than for heifers. While not significant, heifers that received two embryos tended to have lower pregnancy rates than those that received a single embryo (20% vs. 41%); there was no difference in pregnancy rate in cows (50% for two embryos vs. 57% for one embryo). Pregnancy losses between Day 64 and Day 127 occurred in one group only cows receiving two embryos. In that group, pregnancy rate was 50% at Day 64 but 17% at Day 127. Overall, there was no difference in pregnancy rates at day 127 between cows and heifers, but recipients that received two embryos (17% for cows and 20% for heifers) had lower pregnancy rates (P &lt; 0.03) than recipients that received one embryo (57% for cows and 41% for heifers). Only one animal, a cow, had twin fetuses at day 127. In conclusion, unilateral transfer of two embryos failed to improve pregnancy rates of dairy cattle exposed to heat stress. The fact that fetal loss occurred sooner for heifers than for cows points out the importance of uterine capacity as a limiting factor for maintenance of fetal development of two conceptuses. The suitability of timed embryo transfer was evident from the high pregnancy rates achieved with crossbreed females that received a single embryo. This work was supported by BARD Grant No. US-1551-14, USDA TSTAR Grant No. 2004-14135-14715, Grant No. 2001-12101-11318 from the USDA-IFAFS Program, and CAPES Grant No. 134202-1).

Pregnancy rates following timed embryo transfer with fresh or vitrified in vitro produced embryos in lactating dairy cows under heat stress conditions

Timed embryo transfer (TET) using in vitro produced (IVP) embryos without estrus detection can be used to reduce adverse effects of heat stress on fertility. One limitation is the poor survival of IVP embryos after cryopreservation. Objectives of this study were to confirm beneficial effects of TET on pregnancy rate during heat stress as compared to timed artificial insemination (TAI), and to determine if cryopreservation by vitrification could improve survival of IVP embryos transferred to dairy cattle under heat stress conditions. For vitrified embryos (TET-V), a three-step pre-equilibration procedure was used to vitrify excellent and good quality Day 7 IVP Holstein blastocysts. For fresh IVP embryos (TET-F), Holstein oocytes were matured and fertilized; resultant embryos were cultured in modified KSOM for 7 days using the same method as for production of vitrified embryos. Excellent and good quality blastocysts on Day 7 were transported to the cooperating dairy in a portable incubator. Nonpregnant, lactating Holsteins ( n=155) were treated with GnRH (100 μg, i.m., Day 0), followed 7 days later by prostaglandin F 2α (PGF 2α, 25 mg, i.m.) and GnRH (100 μg) on Day 9. Cows in the TAI treatment ( n=68) were inseminated the next day (Day 10) with semen from a single bull that also was used to produce embryos. Cows in the other treatments ( n=33 for TET-F; n=54 for TET-V) received an embryo on Day 17 (i.e. Day 7 after anticipated ovulation and Day 8 after second GnRH treatment). The proportion of cows that responded to synchronization based on plasma progesterone concentrations on Day 10 and Day 17 was 67.7%. Pregnancy rate for all cows on Day 45 was higher ( P<0.05) in the TET-F treatment than for the TAI and TET-V treatments (19.0±5.0, 6.2±3.6, and 6.5±4.1%). For cows responding to synchronization, pregnancy rate was also higher ( P<0.05) for TET-F than for other treatments (26.7±6.4, 5.0±4.3, and 7.4±4.7%). In the TET-F treatment group, cows producing more milk had lower ( P<0.05) pregnancy rates than cows producing less milk. In conclusion, ET of fresh IVP embryos can improve pregnancy rate under heat stress conditions, but pregnancy rate following transfer of vitrified embryos was no better than that following TAI.

Adverse impact of heat stress on embryo production: causes and strategies for mitigation

The production of embryos by superovulation is often reduced in periods of heat stress. The associated reduction in the number of transferable embryos is due to reduced superovulatory response, lower fertilization rate, and reduced embryo quality. There are also reports that success of in vitro fertilization procedures is reduced during warm periods of the year. Heat stress can compromise the reproductive events required for embryo production by decreasing expression of estrus behavior, altering follicular development, compromising oocyte competence, and inhibiting embryonic development. While preventing effects of heat stress can be difficult, several strategies exist to improve embryo production during heat stress. Among these strategies are changing animal housing to reduce the magnitude of heat stress, utilization of cows with increased resistance to heat stress (i.e., cows with lower milk yield or from thermally-adapted breeds), and manipulation of physiological and cellular function to overcome deleterious consequences of heat stress. Effects of heat stress on estrus behavior can be mitigated by use of estrus detection aids or utilization of ovulation synchronization treatments to allow timed embryo transfer. There is some evidence that embryonic survival can be improved by antioxidant administration and that pharmacological treatments can be developed that reduce the degree of hyperthermia experienced by cows exposed to heat stress.

Efficacy of Timed Embryo Transfer with Fresh and Frozen In Vitro Produced Embryos to Increase Pregnancy Rates in Heat-Stressed Dairy Cattle

Our objective was to determine whether pregnancy rates in heat-stressed dairy cattle could be enhanced by timed embryo transfer of fresh (nonfrozen) or frozenthawed in vitro-derived embryos compared to timed insemination. Ovulation in Holstein cows was synchronized by a GnRH injection followed 7 d later by PGF2α and a second treatment with GnRH 48h later. Control cows (n=129) were inseminated 16h (d 0) after the second GnRH injection. On d 7, a fresh (n=133) or frozen-thawed (n=142) in vitro-derived embryo was transferred to cows assigned for timed embryo transfer after categorizing the corpus luteum by palpation per rectum as 3 (excellent), 2 (good or fair), 1 (poor), and 0 (nonpalpable). Response to the synchronization treatment, determined by plasma progesterone concentration (ng/ml) ≤1.5 on d 0 and ≥2.0 on d 7, was 76.2%. Mean plasma progesterone concentration on d 7 increased as the quality of corpus luteum improved from category 0 to 3. Concentrations of progesterone in plasma were elevated (≥2.0ng/ml) at 21 d in 64.7 (fresh embryo), 40.3 (frozen embryo), and 41.4 ±0.1% (timed insemination) of cows, respectively. Cows that received a fresh embryo had a greater pregnancy rate at 45 to 52 d than did cows that received a frozen-thawed embryo or timed insemination (14.3>4.8, 4.9 ± 2.3%). Body condition (d 0) of cows influenced the pregnancy rate and plasma progesterone concentrations. In summary, timed embryo transfer with fresh in vitro-produced embryos in heat-stressed dairy cattle improved pregnancy rate relative to timed insemination.

Antithyroid antibodies do not affect pregnancy outcome in women undergoing assisted reproduction.

The purpose of this study was to determine the association of antibodies to thyroglobulin and thyroid peroxidase and pregnancy outcome in women undergoing assisted reproductive techniques. The study included three centres and retrospectively evaluated patient sera for antithyroid antibodies, then related the results to pregnancy outcome. Enzyme-linked immunosorbant assays for thyroglobulin and thyroid peroxidase antibodies were performed using two different commercially available kits. Controls included 200 healthy women of reproductive age. Women (n = 873) who were undergoing assisted reproductive techniques for pelvic adhesions, endometriosis, ovarian dysfunction, or unexplained/other were included. All women utilized a standard regimen of gonadotrophin releasing hormone agonist down-regulation followed by ovulation induction. Embryos were cultured for 3 days, at which time embryo transfer occurred. Statistical analysis utilized the two-tailed Fisher's exact test. Antithyroid antibodies were positive in 143 of 873 (16.4%) women undergoing assisted reproductive techniques while 29 of 200 (14.5%) normal controls were positive (not significant). Deliveries were achieved in 396 of 730 (54.2%) of women who had no thyroid antibodies versus 78 of 143 (54.5%) of women who had thyroid antibodies (not significant). No difference in biochemical pregnancies (not significant) or clinical pregnancy losses (not significant) were detected. Antithyroid antibodies were found no more frequently in women undergoing assisted reproductive techniques than in normal controls. There were no differences in pregnancy outcome based on antithyroid antibody positivity in women undergoing assisted reproductive techniques. These data do not support the testing or treatment for antithyroid antibodies of women undergoing assisted reproductive techniques.

High success with gestational carriers and oocyte donors using synchronized cycles

Our purpose was to determine if exogenous hormone stimulation of the endometrium improves the implantation and pregnancy rate with a host uterus or gestational carrier. Nine couples underwent 14 embryo transfer cycles with a gestational carrier. The median age was 31.3 years for the infertile women donating the oocytes and 36.6 years for the gestational carriers. Eight pregnancies were achieved documented by ultrasound confirmation of cardiac activity. The pregnancy rate was 64.3%, with 1.6 cycles per patient. We show that synchronized exogenous hormone stimulation of the gestational carrier improves the success rate of embryo transfer. When the success rate is compared to a timed embryo transfer with the gestational carrier's natural cycle, from previous reported studies, exogenous hormonal stimulation appears to enhance endometrial receptivity and improve the implantation rate.