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.     

The usefulness of a single measurement of insulin-like growth factor-1 as a predictor of embryo yield and pregnancy rates in a bovine MOET program

The objective was to determine if a single measurement of plasma insulin-like growth factor-1 (IGF-1) could predict the number of viable embryos obtained from donors and the likelihood of pregnancy in recipients in multiple ovulation and embryo transfer (MOET) programs in cattle. The embryo yields from 101 embryo recoveries were examined in maiden Holstein heifers (n=75) and multiparous Holstein cows (lactating cows n=20, dry cows n=6). Donors were super stimulated with FSH and embryo recovery was done non-surgically 7 days after artificial insemination. Embryos were classified according to the IETS criteria. Pregnancy rates in 100 maiden Holstein heifer recipients were analysed. Recipients were on day 7+/-1 of the estrous cycle at transfer. Pregnancy diagnosis was carried out at day 30 (PD 30) and rechecked at day 60 (PD 60) after transfer. Blood samples from coccygeal vessels taken at the time of embryo recovery (donors) and transfer (recipients) were analysed for IGF-1, insulin, beta-hydroxybutyrate (beta-OHB), non-esterified fatty acids (NEFA), urea and cholesterol. There was a negative correlation between the number of viable embryos and insulin (r=-0.33, P=0.025) in donor heifers. In donor cows, the number of viable embryos was correlated with IGF-1 (r=0.43, P=0.028) and cholesterol (r=-0.43, P=0.027). In recipients, PD30 and PD 60 were not affected by any of the circulating parameters analysed. Insulin, IGF-1 and cholesterol only explained 8.9, 13.9 and 15.8% of the variation in the production of viable embryos, respectively. Several factors affect MOET programs and under the circumstances of the present study the usefulness of hormonal and metabolic profiles as predictors of the outcome of this biotechnology was limited.     

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 microg 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.     

Influence of summer heat stress on pregnancy rates of lactating dairy cattle following embryo transfer or artificial insemination

Lactating Holstein cows were used to determine if pregnancy rate from embryo transfer (n = 113) differed from contemporary control cows (n = 524) that were artificially inseminated (AI). Holstein heifers (n = 55) were superovulated with FSH-P (32 mg total) and inseminated artificially during estrus and subsequently managed under shade structures. On Day 7 post estrus, embryos were recovered, and primarily excellent to good quality embryos (90.3%) were transferred to estrus-synchronized lactating cows. Cows were managed under conditions of exposure to summer heat stress. Pregnancy status was determined by milk progesterone concentrations at Day 21 and palpation per rectum at 45 to 60 d post estrus. Pregnancy rates of cows presented for AI (Day 21, 18.0%; Days 45 to 60, 13.5%) were typical for lactating cows inseminated during periods of summer heat stress in Florida. Pregnancy rates of embryo recipient cows were higher (P<0.001) than those of control cows (Day 21, 47.6%; Days 45 to 60, 29.2%). Summer heat stress had no adverse effect on heifer superovulatory response, but it increased (P<0.05) the incidence of retarded embryos (相似文献   

Establishment of pregnancies after serial dilution or direct transfer by vitrified equine embryos

Experiments were conducted to determine viability of equine embryos in vivo after vitrification. In a preliminary study (Experiment 1), embryos were exposed in three steps to vitrification solutions containing increasing concentrations of ethylene glycol and glycerol (EG/G); the final vitrification solution was 3.4 M glycerol + 4.6 M ethylene glycol in a base medium of phosphate-buffered saline. Embryos were warmed in a two-step dilution and transferred into uteri of recipients. No pregnancies were observed after transfer of blastocysts >300 microm (n = 3). Transfer of morulae or blastocysts < or = 300 microm resulted in four embryonic vesicles (4/6, 67%). In a second experiment, embryo recovery per ovulation was similar for collections on Day 6(28/36, 78%) versus Days 7 and 8(30/48, 62%). Embryos < or = 300 and >300 microm were vitrified, thawed and transferred as in Experiment 1. Some embryos < or = 300 microm were also transferred using a direct-transfer procedure (DT). Embryo development rates to Day 16 were not different for embryos < or = 300 microm that were treated as in Experiment 1(10/22, 46%) or transferred by DT (16/26, 62%). Embryos > 300 microm (n = 19) did not produce embryonic vesicles.     

Comparison of embryo quality in high-yielding dairy cows, in dairy heifers and in beef cows

The purpose of this study was to compare embryo quality of lactating Holstein Friesian cows (LHFC), non-lactating Holstein Friesian heifers (NLHFH) and Belgian Blue beef cows (BB) and to identify factors that are associated with embryo quality in LHFC and NLHFH. After superovulation and embryo recovery at Day 7, embryos (n=727 from 47 LHFC, 27 NLHFH and 50 BB) were scored morphologically for quality, colour and developmental stage. Blood samples and data concerning parity, age, milk production and management were collected. Data were compared univariably between the three groups. A multivariable regression model was built with quality and colour of the LHFC and NLHFH embryos as dependent variables. Only 13.1% of LHFC embryos were categorized as excellent compared to 62.5% and 55.0% of the embryos in NLHFH and BB, respectively. Almost none of the NLHFH or BB embryos displayed a dark appearance of the cytoplasm compared to 24.1% of the LHFC embryos. Only 4% of all LHFC embryos reached blastocyst stage compared to 23.2% and 17.3% in NLHFH and BB. Based on the multivariable regression analysis, "physiological status" (lactating or not) together with the serum total protein concentration of LHFC and NLHFH, was significantly associated with embryo quality and colour. Thus, LHFC display an inferior embryo quality compared to NLHFH and BB. Producing milk or not seems to be significantly associated with embryo quality. Therefore, reduced embryo quality on Day 7 following AI, could be an important factor in the subfertility problem in modern high-yielding dairy cows.     

Plasma progesterone profiles and factors affecting embryo-fetal mortality following embryo transfer in dairy cattle

The relationship between plasma progesterone (P4) levels and embryo survival, and the value of P4 profiles for the selection of cattle embryo transfer recipients is still a matter of controversy. This study reports a comparison between lactating cows and heifers (n = 407) from a single dairy herd, after transfer of either fresh or frozen-thawed good quality embryos, of their ability to sustain embryo-fetal development to term. Plasma P4 concentrations on the day of estrus (Day 0 = D0), Day 4, Day 7 and on Day 21 were measured and related to embryo survival. Plasma P4 levels on Days 0, 4 and 7 were similar in recipients later found pregnant or open. Plasma P4 levels on Day 7 were significantly higher (P < 0.01) in heifers than in cows, but they were similar in pregnant and nonpregnant heifers and in pregnant and nonpregnant cows. Pregnancy rates for fresh and frozen-thawed embryos were higher in heifers than in cows, but the differences did not reach significance. However, the overall late embryonic mortality was significantly higher (P < 0.01) and the calving rate for frozen-thawed embryos was significantly lower (P < 0.05) in cows than in heifers. As expected, plasma P4 on Day 21 was significantly higher (P < 0.001) in pregnant than in nonpregnant recipients, but there was no difference between pregnant cows and pregnant heifers. Plasma P4 levels on Day 7 of recipients presumed pregnant on Day 21 and later found pregnant or nonpregnant were similar, but plasma P4 levels on Day 21 were significantly higher (P < 0.001) in pregnant than in nonpregnant recipients. The results of this study suggest that plasma P4 levels until the day of transfer, except for the rejection of recipients with abnormal luteal function, are of limited practical use for embryo transfer recipient selection. However, in lactating cows low plasma P4 values on Day 7 might negatively affect embryo survival, while in heifers this effect is not noticeable. Lactating cows are more prone to embryo loss than heifers, especially in the case of frozen-thawed embryos; this is associated with a lower competence of the corpus luteum at Day 7.     

Effect of recombinant bovine somatotropin on superovulatory response and recipient pregnancy rates in a commercial embryo transfer program

Recombinant bovine somatotropin (rbST) has been shown to increase follicular growth in cattle and some studies have demonstrated an increase in superovulatory response for rbST-treated cows. Pregnancy rates have also been shown to increase when rbST was administered around the time of insemination or prior to embryo transfer. The application of rbST for the purpose of increasing superovulatory responses of donor cows and increasing pregnancy rates of recipient heifers was tested in a commercial embryo transfer program. In Experiment 1, embryo donor cows (n = 56) underwent three cycles of control superovulation (two before and one after weaning) and subsequently underwent up to four additional superovulations while being treated with either rbST (500 mg sustained-release rbST; Posilac, Monsanto, St. Louis, MO; n = 28) or excipient (control; n = 28) once every 14 days. In Experiment 2, lactating embryo donor cows (n = 37) underwent a control superovulation and then underwent a superovulation while lactating and being treated with either rbST (n = 16) or excipient (n = 21). In Experiment 3, embryo recipient heifers that were being implanted with either in vitro or in vivo produced embryos were treated with either rbST (n = 146) or excipient (n = 143) at the time of embryo transfer. Treatment of non-lactating (Experiment 1) or lactating (Experiment 2) donor cows with rbST during repeated superovulation did not affect the number of corpora lutea, the sum of transferable embryos, degenerate embryos, and unfertilized oocytes, or the number of transferable embryos. Treatment of recipient heifers with rbST (Experiment 3) did not affect pregnancy rates for either in vitro or in vivo produced embryos. We conclude that superovulatory response and pregnancy rates (respectively) are similar to control for rbST-treated cows undergoing repeated superovulations and rbST-treated recipient heifers treated at the time of embryo transfer.     

Embryo loss in cattle between Days 7 and 16 of pregnancy

Embryo loss between embryonic Days 7 and 16 (Day 0 = day of IVF) in nonlactating cattle, Bos taurus, was analyzed using transfer of 2449 (in groups of 3 to 30) in vitro-produced (IVP) blastocysts. In 152 transfers, pregnancy losses attributable solely to recipient failings amounted to between 6% (beef heifers) and 16% (parous dairy cows), of which 3% were caused by uterine infections. Neither season, year, nor the age of the embryos on retrieval affected pregnancy rates. The latter observation indicated that the reason that a recipient failed to retain embryos was already present at the time of transfer. Notably, the proportion of embryos recovered decreased (P = 0.03) as more embryos were transferred, particularly at later stages (Day 14, P < 0.01). The average length of embryos decreased by approximately 5% for every additional embryo transferred (P < 0.0001). These effects may be linked to embryonic migration. Embryo mortality inherent to the embryo during the second week of pregnancy was 24%. Additionally, 9% of Day 14 embryos were of inferior quality, as they did not contain an epiblast. Combining embryo and recipient causes but excluding infection effects, embryonic loss of IVP embryos during the second week of pregnancy amounted to 26% (heifers) or 34% (parous dairy cows). The length of embryos doubled every day between Days 9 and 16, with a 4.4-fold range in sizes representing two thirds of the variation in length. Embryos retrieved from heifers were twice the size of those incubated in parous cows (P < 0.0001), indicating faster embryonic development/trophoblast proliferation in heifers. Whereas season did not affect embryo recoveries, length was lower (50%) in winter (winter-autumn, P < 0.05; winter-spring, P < 0.001). Lastly, transuterine migration in cattle, when transferring multiple embryos, commenced at Day 14 (4%) and had occurred in all recipients by Day 16 (38% of embryos found contralaterally).     

The effects of cryopreservation and transfer on embryonic development in the common marmoset monkey, Callithrix jacchus

Embryos were collected at the 4-10-cell stage from the oviducts (Day 4; Day 1 = ovulation) or as morulae (Day 7) from the uterus of marmosets and frozen in 1.5 M-DMSO (Days 4 and 7) or 1.0 M-glycerol (Day 4 only), using a slow freezing and thawing technique. Of 22 Day-4 embryos frozen in DMSO, 18 were recovered and 16 of these were transferred to 10 synchronized recipients; 7 recipients became pregnant compared with all 7 control recipients receiving 10 unfrozen embryos. Fifteen frozen-thawed morulae were transferred to 9 Day-6 recipients; the pregnancy rate (55.6%) was lower than for control embryos (85.7%). Embryos frozen in glycerol suffered severe osmotic stress during glycerol addition and removal. Of 8 recipients, 3 (37.5%) became pregnant but only one fetus was carried to term. These results on embryo collection, freezing and transfer in the marmoset have important implications for developing improved methods for freezing human embryos and the breeding of endangered primates.     

Conception rates after artificial insemination or embryo transfer in lactating dairy cows during summer in Florida

The objective was to compare conception rates to embryo transfer relative to AI, during summer heat stress, in lactating dairy cows. Holstein cows (n = 180; 50 to 120 d postpartum) were allocated randomly to 1 of 3 groups: artificial insemination (AI, n = 84), embryo transfer using either embryos collected from superovulated donors (ET-DON, n = 48), or embryos produced in vitro (ET-IVF, n = 48). Embryos from superovulated donors were frozen in 10% glycerol and were rehydrated in a 3-step procedure, in decreasing concentrations of glycerol in a sucrose medium before transfer. Embryos produced in vitro were frozen in 1.5 M ethylene glycol, thawed and transferred without rehydration. Blood samples were collected from AI and ET recipients on Days 0, 7 and 22 for measurement of progesterone in plasma. Conception rate was estimated for the three groups at Day 22 (progesterone > 1 ng/mL) and confirmed at Day 42 by palpation per rectum. Conception rate estimates at Day 22 did not differ among groups (AI, 60.7%; ET-DON, 60.4%; ET-IVF, 54.2%), but conception rates at Day 42 differed (AI, 21.4%; ET-DON, 35.4%; ET-IVF, 18.8%; AI versus ET: P > 0.10 and ET-DON versus ET-IVF: P < 0.05). In cows considered pregnant at 22 d but diagnosed open at 42 d, the interestrous intervals were 28.8 +/- 2.2, 35.2 +/- 3.5 and 31.6 +/- 2.9 d, respectively, for AI, ET-DON and ET-IVF groups. Transfer of embryos collected from nonheat-stressed superovulated donors significantly increased conception rates in heat stressed dairy cattle. However, transfer of IVF-derived embryos had no advantage over AI. Where appropriate mechanisms are in place to attenuate the effects of heat stress, embryo transfer using frozen-thawed donor embryos increases conception rates.     

Factors affecting pregnancy rates and early embryonic death after equine embryo transfer

In the present study, 638 embryo transfers conducted over 3 yr were retrospectively examined to determine which factors (recipient, embryo and transfer) significantly influenced pregnancy and embryo loss rates and to determine how rates could be improved. On Day 7 or 8 after ovulation, embryos (fresh or cooled/transported) were transferred by surgical or nonsurgical techniques into recipients ovulating from 5 to 9 d before transfer. At 12 and 50 d of gestation (Day 0 = day of ovulation), pregnancy rates were 65.7% (419 of 638) and 55.5% (354 of 638). Pregnancy rates on Day 50 were significantly higher for recipients that had excellent to good uterine tone or were graded as "acceptable" during a pretransfer examination, usually performed 5 d after ovulation, versus recipients that had fair to poor uterine tone or were graded "marginally acceptable." Embryonic factors that significantly affected pregnancy rates were morphology grade, diameter and stage of development. The incidence of early embryonic death was 15.5% (65 of 419) from Days 12 to 50. Embryo loss rates were significantly higher in recipients used 7 or 9 d vs 5 or 6 d after ovulation. Embryos with minor morphological changes (Grade 2) resulted in more (P<0.05) embryo death than embryos with no morphological abnormalities (Grade 1). Between Days 12 and 50, the highest incidence of embryo death occurred during the interval from Days 17 to 25 of gestation. Embryonic vesicles that were imaged with ultrasound during the first pregnancy exam (5 d after transfer) resulted in significantly fewer embryonic deaths than vesicles not imaged until subsequent exams. In the present study, embryo morphology was predictive of the potential for an embryo to result in a viable pregnancy. Delayed development of the embryo upon collection from the donor or delayed development of the embryonic vesicle within the recipient's uterus was associated with a higher incidence of pregnancy failure. Recipient selection (age, day after ovulation, quality on Day 5) significantly affected pregnancy and embryo loss rates.     

Ovarian responses and embryo survival in recipient lactating Holstein cows treated with equine chorionic gonadotropin

The objectives of Experiment 1 were to determine a dose of eCG that would increase total luteal volume and plasma progesterone (P4) concentration on estrous cycle Day 7 in cows. The objectives of Experiment 2 were to determine the effects of treating embryo recipient lactating Holstein cows with eCG on pregnancy per embryo transfer (P/ET). In Experiment 1, lactating dairy cows at 63 ± 3 d postpartum (DIM) received no treatment (control, n = 10), or 600 (eCG6, n = 19), or 800 (eCG8, n = 19) IU of eCG 2 d after the start of the ovulation-synchronization protocol, Day -8 (Day -10 GnRH, Day -3 PGF_2α, Day 0 GnRH). Blood was sampled on Days -10, -8, -3, 0, 7, and 14 for P4 concentration. Ovaries were examined by ultrasound on Days -10, -3, 0, and 7. In Experiment 2, lactating dairy cows were paired according to parity and previous insemination (0 or > 1 insemination) and assigned to receive 800 IU of eCG (eCG8, n = 152) 2 d after the start of the ovulation-synchronization protocol (Day -10 GnRH, Day -3 PGF_2α, Day 0 GnRH) or to receive no treatment (control, n = 162). Blood was sampled on Days -10, -3, 0, 7, and 14 for determination of P4 concentration. Ovaries were examined by ultrasound on Days -10, -3, and 7, and cows with a CL > 20 mm in diameter on Day 7 received an embryo. In Experiment 1, P4 concentration on Day 7 was higher (P < 0.05) for eCG8 cows (2.3 ± 0.3 ng/mL) compared with control (1.2 ± 0.3 ng/mL) and eCG6 (1.1 ± 0.3 ng/mL) cows. In Experiment 2, eCG8 primiparous cows had more (P < 0.01) follicles > 10 mm on Day -3 compared with control primiparous cows (2.5 ± 0.9 vs 1.7 ± 0.5 mm), but multiparous control and eCG8 cows did not differ. A larger (P = 0.03) percentage of control cows received an embryo (87.5 vs 79.1%) compared with eCG8 cows. Among cows that received an embryo, total luteal volume on Day 7 was affected (P = 0.05) by treatment (eCG8 = 8.3 ± 0.4 cm³, control = 6.2 ± 0.4 cm³), but P4 concentration on Day 7 did not differ significantly between treatments. The percentage of cows pregnant 53 d after ET (overall, 24.2%) was not significantly different between control and eCG8 cows. In the current study, no differences in P/ET were observed between control and eCG8 cows and treatment with eCG increased the percentage of cows with asynchronous estrous cycle.     

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 microg, i.m., Day 0), followed 7 days later by prostaglandin F2alpha (PGF2alpha, 25 mg, i.m.) and GnRH (100 microg) 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.     

Survival of DNA-injected cow embryos temporarily cultured in rabbit oviducts

Holstein or Angus cows were superovulated, inseminated with fresh bull semen, and necropsied about 12 h after estimated time of ovulation. Ova were centrifuged at 15,600 G for 3 to 8 min to reveal pronuclei. In Experiment 1, pronuclear bovine embryos were transferred to ligated or unligated oviducts of 1-d pseudopregnant rabbits for 7 d; 30 of 32 embryos were recovered from ligated oviducts but only 2 of 26 from oviducts and uterine horns of unligated oviducts. In Experiment 2, a Rous sarcoma virus-chloramphenicol acetyl transferase fusion gene was injected into one pronucleus of about half of 404 fertilized bovine ova, using a micromanipulator and interference contrast optics. Injected and noninjected embryos were then transferred to opposite ligated rabbit oviducts. Embryos were recovered after 7, 8 or 9 d. Of 120 centrifuged but ininjected embryos recovered from rabbit oviducts, 66 (55%) were in the morula to hatching blastocyst stage of development. Of 105 embryos centrifuged and injected with foreign DNA, 55 (52%) were in the morula to hatching blastocyst stage. In Experiment 3, centrifuged bovine embryos, noninjected or DNA-injected, were cultured in rabbit oviducts for 7 d then transferred nonsurgically to the uterus of recipient cows. Embryos were also flushed from superovulated cows 8 d after estrus and transferred directly to recipient cows. After 7 d, the uterus of recipient cows was flushed nonsurgically to recover embryos. The proportion of transferred embryos recovered with normally elongated trophoblastic membranes and the proportion of recipient cows with developing embryos were 14 of 25 DNA-injected embryos, 5 of 8 cows; 6 of 15 centrifuged but noninjected embryos, 4 of 6 cows; and 11 of 29 embryos transferred directly, 5 of 8 cows. Results indicate that bovine embryos can be cultured in rabbit oviducts and survive after transfer to cow uteri and that injection of foreign DNA may not increase embryonic loss within the first 2 wk after injection.     

Embryonic mortality and the uterine environment in first-service lactating dairy cows

Embryos were collected non-surgically from the tip of one uterine horn of 23 lactating dairy cows on Day 7 of pregnancy. Embryos were classified on the basis of morphological criteria as normal (n = 12) or abnormal (n = 13). Abnormal embryos were further classified as cleavage stage (n = 9) or morula/blastocyst (n = 4). Cows producing an abnormal embryo did not differ in days post partum at oestrus, age or parity from cows producing a normal embryo. Cows with an abnormal morula/blastocyst also did not differ with respect to days post partum at oestrus from cows with abnormal cleavage-stage embryos but cows with an abnormal morula/blastocyst were significantly older and of greater parity than cows with an abnormal cleavage-stage embryo. Hepes-saline-PVP solution (30 ml) was initially infused into the uterine tip, mixed and then withdrawn with a syringe. Analysis of this fluid revealed that the concentrations of glucose, total protein, calcium, magnesium and potassium were significantly higher in the flushings from the uterus of cows with abnormal embryos than from cows with normal embryos and zinc and phosphorus tended to be higher in the uterine flushings of cows with abnormal embryos. Phosphorus, total protein, calcium and magnesium tended to be higher in the flushings from cows with abnormal morulae/blastocysts than from cows with abnormal cleavage-stage embryos. Plasma progesterone did not differ between cows with normal or abnormal embryos or in cows with abnormal morulae/blastocysts or abnormal cleavage-stage embryos. Most embryonic mortality therefore occurred before Day 5 (during cleavage) in these cows.(ABSTRACT TRUNCATED AT 250 WORDS)     

Embryo recovery rate and recipients’ pregnancy rate after nonsurgical embryo transfer in donkeys

Sixty-three embryos were recovered out of 83 estrous cycles (75.9%) and 98 ovulations (64.3%) of five Pantesca jennies, 2 to 5 yr old, naturally mated or artificially inseminated with fresh semen. Embryo recovery rate was influenced by number of ovulations per cycle (133% and 63% for double and single ovulations, respectively), by the day of embryo recovery attempt (12%, 83%, and 75% at Days 7, 8, and 9 after ovulation, respectively), and by the repetition of the embryo recovery attempt on successive cycles (60%, 79%, and 100% for cycles 1 to 7, 8 to 14, and 15 to 24, respectively). All recovered embryos but three were classified as good or excellent. Of 58 nonsurgical embryo transfers to Ragusana jenny recipients, 13 (22.4%), 10 (17.2%), and 9 (15.5%) resulted in a pregnancy at Days 14, 25, and 50, respectively. Recipients’ pregnancy rate was not influenced by the evaluated parameters: embryo quality and age, media employed to wash embryos, days after ovulation of the recipient, experience of the operator. Between 14 and 50 d of pregnancy, 4 of 13 (30.7%) embryos were lost with an influence of the days from ovulation of the recipient: recipients at Days 5 or 6 kept all pregnancies (N = 7), whereas recipients at Days 7 or 8 lost 3 of 4 pregnancies, as one of the two recipients at Day 3. More studies are needed before embryo transfer could be considered a reliable tool to preserve endangered donkey breeds.     

Bovine somatotropin increases embryonic development in superovulated cows and improves post-transfer pregnancy rates when given to lactating recipient cows

Previous studies indicated that the use of bovine somatotropin (bST) in concurrence with a timed artificial insemination (TAI) protocol increased pregnancy rates. However, the mechanisms for such a bST effect on fertility were not clear. Objectives of this study were to determine the effects of bST on fertilization and early embryonic development after cows received a superovulation treatment, test whether embryos recovered from bST-treated cows were more likely to survive after transfer to recipients, and evaluate whether treatment of recipient cows with bST affects pregnancy rates. Lactating (n = 8) and nonlactating (n = 4) Holstein donor cows were superovulated, inseminated at detected estrus and assigned to a nontreated control group or to a treatment group receiving a single injection of bST (500 mg, sc) at insemination. Embryos were nonsurgically flushed 7 days after AI and frozen in ethylene glycol for direct transfer. Embryos derived from bST-treated (bST-embryos) or control (control-embryos) donors were transferred to lactating Holstein recipient cows that received either bST treatment 1 day after estrus (500 mg, sc; bST-recipients) or were untreated controls (control-recipients). Thus, there were four treatment groups: control-embryos/control-recipients (n = 43), bST-embryos/control-recipients (n = 41), control-embryos/bST-recipients (n = 37), and bST-embryos/bST-recipients (n = 60). Pregnancy was determined by palpation per rectum 33-43 days after embryo transfer. Unfertilized ova per flush was less for bST than for control (1.0 +/- 0.9 < 3.7 +/- 0.9; P < 0.04). Percentage of transferable embryos was greater for bST than for control (77.2% > 56.4%; P < 0.01). Number of blastocysts per flush was greater for bST than for control (2.4 +/- 0.7 > 0.4 +/- 0.7; P < 0.04). Pregnancy rates following embryo transfer were 25.6% for control-recipient/control-embryo, 43.2% for bST-recipient/control-embryo, 56.1% for control-recipient/bST-embryo, and 43.3% for bST-recipient/bST-embryo. Transfer of bST-embryos increased pregnancy rates compared with transfer of control-embryos (P < 0.04). An interaction between embryo and recipient treatments (P < 0.05) indicated that treatment of recipient cows with bST increased pregnancy rates as compared to control-recipients that received a control-embryo. However, there was no additive effect when bST-recipients received a bST-embryo. Administration of bST at AI decreased the number of unfertilized ova, increased the percentage of transferable embryos, and stimulated embryonic development to the blastocyst stage. Moreover, bST affected both early embryonic development and recipient components to increase pregnancy rates following embryo transfer.     

Follicular growth, ovulation and embryo recovery in dairy cows given FSH at the beginning or middle of the estrous cycle

Variability in the superovulation response is an important problem for the embryo transfer industry. The objective of this study was to determine whether FSH treatment at the beginning of the cycle would improve the ovulation rate and embryo yield in dairy cows. Twenty-eight postpartum cyclic dairy cows were allocated at random to 4 treatment groups (A, B, C and D). Group A cows (n = 10) received FSH (35 mg) at a decreasing dose, starting on Day 9 (Day 0 = day of estrus) for 5 days followed by PGF(2alpha) (35 mg) on Day 12. Cows assigned to Groups B, C and D (n = 6 cows each, respectively) were given 35 mg FSH at a decreasing dose from Days 2 to 6 followed by PGF(2alpha) on Day 7. Group C and D cows received PRID inserts from Day 3 to Day 7. Cows in Group D additionally received 1000 IU hCG 60 hours after PGF(2alpha) treatment. Ovaries were scanned daily using a real time ultrasound scanner from the beginning of FSH treatment until embryo recovery, to monitor follicular development, ovulation and the number of unovulated follicles. Embryos were recovered from the uterus by a nonsurgical flushing technique 7 days after breeding. There were no differences (P>0.01) in the number of follicles > 10 mm at 48 hours after PGF(2alpha) treatment among the 4 groups. The mean numbers of follicles were 10.6 +/- 1.2, 9.3 +/- 1.3, 12.2 +/- 1.3 and 15.0 +/- 2.9 for Groups A, B, C and D, respectively. A significantly (P<0.001) higher number of ovulations was observed and a larger number of embryos was recovered in Group A than in the other groups. The results of this study indicate that superovulation with FSH at the beginning of the cycle causes sufficient follicular development but results in very low ovulation and embryo recovery rates.