Bovine embryo morphology and evaluation

The following paper briefly reviews the morphology of the bovine embryo and presents a retrospective analysis of bovine embryo transfer results accumulated from April to December of 1982 at a commercial embryo transfer center. Of particular interests were bovine embryo morphology, assessment of embryo quality, and recipient-donor, recipient-embryo synchrony requirements. Embryos were recovered from superovulated donors five to nine days after estrus (estrus = day O). All embryos were individually examined at 200X for cell stage of development and embryo quality. Embryos were nonsurgically transferred to recipients that were within two days of estrous cycle synchrony with the donor. Attempts were made to synchronize estimated developmental age of embryos to the day of the recipient cycle. A high degree of variability was observed in morphological development and embryo quality within and among donors. Embryo recovery in individual donors resulted in a wide range of embryonic cell stages, often differing in estimated developmental ages from 24 to 48 hours. A total of 783 embryos were transferred, resulting in 308 pregnancies. Stage of embryonic development (16-cell through hatched blastocyst) had little effect on pregnancy rates. Embryo quality was a more accurate predictor of success. Embryos of excellent, good, fair and poor categories resulted in 45%, 44%, 27% and 20% pregnancy rates, respectively. Recipient-donor estrous cycle synchrony of two days in either direction did not significantly alter pregnancy rates. However, 88% of 258 pregnancies (584 total transfers) occurred with a +/-1 day recipient-embryo synchrony compared to 74% based on +/-1 day recipient-donor cycle synchrony (P<0.001). Results suggest that transfer of bovine embryos based on synchrony between day of recipient cycle and state of embryonic development provides higher pregnancy rates than transfers based on recipient-donor cycle synchrony.     

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.     

Detrimental effects of high plasma urea nitrogen levels on viability of embryos from lactating dairy cows

High plasma urea nitrogen (PUN) concentrations are associated with decreased fertility in lactating dairy cows. Our objective was to evaluate the quality of embryos flushed from superovulated lactating cows having moderate or high PUN concentrations. Subsequent embryo survival was determined after transfer to recipient heifers with either low or high PUN. Lactating Holstein dairy cows (n = 23; 50-120 days in milk) were randomly assigned to one of two diets designed to result in moderate or high PUN concentrations (15.5 +/- 0.7 and 24.4 +/- 1.0 mg/dl, respectively; P < 0.001) and were fed for 30 days before embryo flushing and recovery. Embryos (n = 94) were evaluated morphologically, frozen and subsequently transferred into synchronized virgin heifers that were fed one of two diets designed to result in either low or high PUN concentrations (7.7 +/- 0.9 and 25.2 +/- 1.5 mg/dl, respectively; P < 0.001; 2 x 2 factorial design). The number, quality and stage of development of recovered embryos were similar for cows with moderate or high PUN. Transfer of embryos from moderate PUN donor cows resulted in a higher pregnancy rate (35%; P < 0.02) than the transfer of embryos from high PUN donor cows (11%). Pregnancy rate was not affected by either recipient diet or the interaction of donor and recipient diets (P > 0.05). These results indicate that high PUN concentrations in lactating dairy cows decrease embryo viability through effects exerted on the oocyte or embryo before recovery from the uterus 7 days after insemination.     

Commercial aspects of bovine embryo transfer

Superovulated beef cows and heifers were nonsurgically collected 6 to 8 days post estrus. Commercial production results for 1976 through 1978 were 4979 pregnancies from 7814 embryos transferred for an overall pregnancy rate of 63%. In 1978, 519 superovulation procedures averaged 9.95 +/- 8.4 (S.D.) ova collected, 8.2 +/- 7.55 ova fertilized, 5.96 +/- 5.37 embryos transferred and 3.63 +/- 5.37 pregnancies per procedure. Embryos were transferred to recipient cows in estrus 12 hr before the donor (-12) the same time (0) or 12 hr after the donor (+12). The +12 group had a significantly lower pregnancy rate (61%, P<.05) than the 0 group (67%) or -12 group (66%). Transfer of early morula stage embryos resulted in a lower pregnancy rate (61%, P<.05) than late morula (67%) early blastocyst (67%) or late blastocyst (71%) stage embryos. A higher pregnancy rate (P<.05) was obtained with embryos of good morphological quality (71%) than with embryos graded fair and poor (55%). The pregnancy rate for embryos transferred nonsurgically was lower (44%) than the pregnancy rate for embryos transferred surgically during the same time period (66%). Pregnancy rates for three operators performing the nonsurgical transfers were 48%, 53%, 28%. No difference in pregnancy rate was found between embryos cultured 24 hr in BMOC-3 at 37C (62%) and embryos transferred the same day as collection (60%). Pregnancy rates for cultured embryos transferred to recipient cows in estrus 12, 24 or 36 hr after the donor were 68%, 62% and 60%, respectively. Embryos recovered on days 6, 7 and 8 were frozen in 1.5M DMSO and stored in liquid nitrogen several days to several weeks. Of 68 embryos frozen, 34 were viable post thaw. Upon transfer to recipient cows, the 34 viable embryos produced 23 confirmed pregnancies.     

Superovulation, collection and transfer of embryos and demi-embryos from Boran(Bos indicus ) cows and heifers

Twenty-three Boran(Bos indicus ) cows and heifers were superovulated with pregnant mare serum gonadotropin (PMSG); a total of four embryos and 4.1 +/- 0.3 (mean +/- SEM) ova per ova-producing donor resulted. Follicle stimulating hormone (FSH-P) was then used to superovulate 49 Boran cows for a total of 106 superovulations, of which 63 (59.4%) produced an average of 3.7 +/- 0.4 (mean +/- SEM) embryos. The embryo production was not influenced by either the season or the number of times(one to five) the cows were superovulated. A higher pregnancy rate was obtained when the selection of Boran recipients was based on their plasma-progesterone values (overall 52.5%, single embryos 63.3%, twin demi-embryos 45.8%) than when they were selected by palpation per rectum only (overall 43.8%, single embryos 50%, twin demi-embryos 36.4%). The twinning rate of twin demiembryos was 62.5%, whereas only single calves were born after transfer of two embryos per recipient. No pregnancies were produced following transfer of twin demi-embryos without zonae pellucidae. Transferring single demi-embryos gave a low pregnancy rate (13.3%). Twelve donor Boran cows (21 superovulations) bred with their fathers resulted in a high rate of early embryonic death; additionally, only 20.9% (overall) of the recipients became pregnant. Estrus synchronization of Boran cows with a progesterone releasing intravaginal device (PRID) for a short period (7 d) combined with one injection of prostaglandin (Day 6) produced a larger number of good quality recipients (70.5%) than using double prostaglandin injections (60%).     

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   

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.     

Production of interferon by red deer (Cervus elaphus) conceptuses and the effects of roIFN-tau on the timing of luteolysis and the success of asynchronous embryo transfer

The role of interferon in early pregnancy in red deer was investigated by (a) measuring production of interferon by the conceptus, (b) testing the anti-luteolytic effect of recombinant interferon-tau in non-pregnant hinds, and (c) treatment of hinds with interferon after asynchronous embryo transfer. Blastocysts were collected from 34 hinds by uterine flushing 14 (n = 2), 16 (n = 2), 18 (n = 8), 20 (n = 13) or 22 (n = 9) days after synchronization of oestrus with progesterone withdrawal. Interferon anti-viral activity was detectable in uterine flushings from day 16 to day 22, and increased with duration of gestation (P < 0.01) and developmental stage (P < 0.01). When interferon-tau was administered daily between day 14 and day 20 to non-pregnant hinds to mimic natural blastocyst production, luteolysis was delayed by a dose of 0.2 mg day(-1) (27.3 +/- 1.3 days after synchronization, n = 4 versus 21 +/- 0 days in control hinds, n = 3; P < 0.05). Interferon-tau was administered to hinds after asynchronous embryo transfer to determine whether it protects the conceptus against early pregnancy loss. Embryos (n = 24) collected on day 6 from naturally mated, superovulated donors (n = 15) were transferred into synchronized recipients on day 10 or day 11. Interferon-tau treatment (0.2 mg daily from day 14 to 20) increased calving rate from 0 to 64% in all recipients (0/11 versus 7/11, P < 0.005), and from 0 to 67% in day 10 recipients (0/8 versus 6/9, P < 0.01). The increased success rate of asynchronous embryo transfer after interferon-tau treatment in cervids may be of benefit where mismatched embryo-maternal signalling leads to failure in the establishment of pregnancy.     

Influence of environmental temperature on reproductive performance of bovine embryo donors and recipients in the southwest region of the United States

Superovulation and embryo transfer records on performance of embryo donor (n = 3,908; beef 99%, dairy 1%) and recipient (n = 19,936; beef 92%, dairy 8%) cattle from a commercial transfer unit were analyzed for environmental effects. Embryos (n = 42,428) were recovered on Days 5 to 8 postestrus from superovulated donors. Numbers of ova, fertilized ova and embryos of transferable quality were recorded. Transferable embryos were classified according to stage of development and morphological quality. Embryos (n = 19,936) were transferred nonsurgically. Responses were modeled with maximum-likelihood procedures using log-linear functions of independent variables and ANOVA. Fluctuations in daily maximum temperature (1 to 43 degrees C), for Days 0 to 7 of embryo development, had no effect on distribution of embryos classified as good (48%), fair (40%) and poor (12%). Temperature did not affect the percentage of donors flushed with recoverable ova (89%), mean number of ova (12.2 +/- 0.3), fertilization rate (76%) or percentage of transferable embryos (57%). Recipient pregnancy rate (56%) was not affected by mean maximum temperature for Days 0 to 10 posttransfer. Interactions between temperature and breed type (dairy vs beef), parity (cow vs heifer), or lactational status (lactating vs dry) on pregnancy rate were recorded. Elevated environmental temperature does not appear to adversely affect reproductive responses of donor and recipient cattle intensely managed in a commercial transfer unit.     

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 100ng/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 24h to determine interferon-tau (IFN-tau) secretion. There was no effect of IGF-1 on embryo recovery rate, embryo length or IFN-tau 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-tau 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-tau 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.     

Non-surgical embryo transfer in cattle

Embryos collected surgically from donors superovulated with PMSG and synchronized with either prostaglandin F(2)alpha or progestagen impregnated sponges were transferred non-surgically to prostaglandin or progestagen synchronized recipients. One embryo was transferred to the uterine horn ipsilateral to the corpus luteum either through a flexible catheter introduced through a steel tube and passed to the uterine tip, or through a Cassou inseminating gun passed approximately 6 cm into the horn. Of 16 recipients receiving 5 or 6 day old embryos through the catheter (1976), 6 (38%) were palpated pregnant at 42 days and 4 (25%) subsequently calved. Of 16 recipients receiving 7 or 8 day old embryos through the straw and 16 through the catheter (1977), 10 (63%) and 3 (19%), respectively, were palpated pregnant (P<0.05) and 8 (50%) and 3 (19%), respectively, had normal embryos at slaughter 4 to 29 days after palpation (P reverse similar0.10 ). Forty 7 to 9 day old embryos were transferred through the straw in 1978. Eighteen (45%) of the recipients were palpated pregnant and 16 (40%) had normal embryos at slaughter 98 to 168 days after palpation. The success of the transfers in 1978 was affected by embryo quality [good vs poor embryos; 64% vs 22% recipients pregnant (P<0.01) and 59% vs 17% embryos surviving to slaughter (P<0.05)]. Also, in 1978, pregnancy rate was affected by the time taken to transfer the embryo with the highest rate achieved with the fastest transfers (P<0.10, b = -0.47). Injection of Indomethacin near the time of transfer, synchronization between donor and recipient onset of estrus and embryo age did not affect pregnancy rates. The pregnancy rate achieved after the transfer of good quality embryos by the straw technique was equal to that expected from surgical techniques.     

Plasma concentrations of luteinizing hormone and progesterone during superovulation of dairy cows using follicle stimulating hormone or pregnant mare serum gonadotrophin

Eighteen lactating Holstein cows were randomly divided into three groups of equal size. Six cows were not superovulated; the remaining cows were superovulated using either FSH-P or PMSG beginning on Day 12 of the estrous cycle (day of ovulation = Day 0). Animals treated with FSH-P were injected intramuscularly (i.m.) with 4 mg FSH-P every 12 h for 5 d. PMSG was administered i.m. as a single injection of 2350 IU. Cloprostenol (PG, 500 ug) was injected i.m. 56 and 72 h after commencement of treatment and at the same time in the cycle of controls. All cows were inseminated 56, 68 and 80 h after the first PG injection. Blood samples (5 ml) were collected daily and every 15 min for a period of 9 h on Days -1, 0, 2, 8 and 10, with continuous blood sampling at 15-min intervals during Days 3 to 6. Ovulation rate was 27.7 +/- 8.22 in animals treated with PMSG, and 8.0 +/- 3.2 embryos per donor were recovered. In the FSH group, ovulation rate was 8.3 +/- 1.48 and 3.0 +/- 1.1 embryos per donor were recovered. Progesterone concentrations were similar in all three groups until the onset of the LH surge, when progesterone concentrations were greater (P<0.05) in animals of the PMSG group. After the preovulatory LH surge, concentrations of progesterone started increasing earlier (44 h) in cows treated with PMSG, followed by FSH-treated cows (76 h) and controls (99 h). The LH surge occurred earlier (P<0.05) in PMSG-treated cows (37 h after first PG treatment), than in animals treated with FSH-P (52 h) or controls (82 h). In animals treated with FSH-P, the magnitude of the preovulatory LH surge (24.2 +/- 1.02 ng/ml) was higher (P<0.05) than in the other two groups (PMSG = 17.1 +/- 2.04 ng/ml; control, 16.7 +/- 1.24 ng/ml). Superovulation with FSH-P or PMSG did not affect either mean basal LH concentration, frequency or amplitude of LH pulses during Days -1, 0, 2, 3, presurge periods, or Days 8 and 10 post-treatment. At ovariectomy, 8 d post-estrus, more follicles > 10 mm diam. were observed in the ovaries after treatment with PMSG (8.5 +/- 5.66) than after treatment with FSH-P (0.7 +/- 0.42) (P<0.05). Maximum concentrations of PMSG were measured 24 h after administration. Following this peak, PMSG levels declined with two slopes, with half-lives of 36 h and 370 h.     

Development and survival after transfer of cow embryos cultured from 1-2-cells to morulae or blastocysts in rabbit oviducts or in a simple medium with bovine oviduct epithelial cells

This study compares development of bovine 1-2-cell embryos in bovine oviduct epithelial cell co-culture (Group EC) with a glucose- and serum-free simple medium (CZB), or after surgical transfer to ligated oviducts of rabbits (Group RO). Embryos were surgically collected from superovulated donor cows 40-48 h after the beginning of oestrus and randomly distributed between the two groups. Embryos were cultured or incubated for 5 days. In Exp. 1, embryo quality scores and total numbers of cells in the two groups were compared. In Exp. 2, pairs of similarly treated morulae were transferred to each of 10 or 12 recipients in the Groups RO and EC, respectively. Total cell counts per embryo in both groups averaged 52 (P greater than 0.05), and the in-vitro culture system was equivalent to the rabbit oviducts in promoting embryo development for all characteristics measured. Embryo survival, as determined by ultrasound between Days 39 and 43 after oestrus, in 13 ideal recipients was 57% for embryos in Group EC and 58% for embryos Group RO. None of the 9 less desirable recipients was pregnant for either group. These results establish that cattle zygotes can develop to morulae in culture with bovine oviduct epithelial cells in a simple medium and can produce normal pregnancy rates.     

Inheritance of resistance of bovine preimplantation embryos to heat shock: relative importance of the maternal versus paternal contribution

Brahman preimplantation embryos are less affected by exposure to heat shock than Holstein embryos. Two experiments were conducted to test whether the ability of Brahman embryos to resist the deleterious effects of heat shock was a result of the genetic and cellular contributions from the oocyte, spermatozoa, or a combination of both. In the first experiment, Brahman and Holstein oocytes were collected from slaughterhouse ovaries and fertilized with spermatozoa from an Angus bull. A different bull was used for each replicate to eliminate bull effects. On day 4 after fertilization, embryos >or= 9 cells were collected and randomly assigned to control (38.5 degrees C) or heat shock (41 degrees C for 6 hr) treatments. The proportion of embryos developing to the blastocyst (BL) and advanced blastocyst (ABL; expanded and hatched) stages was recorded on day 8. Heat shock reduced the number of embryos produced from Holstein oocytes that developed to BL (P < 0.001, 55.6 +/- 4.2% vs. 29.8 +/- 4.2%) and ABL (P < 0.01, 37.7 +/- 3.6% vs. 12.2 +/- 3.6%) on day 8 as compared to controls. In contrast, heat shock did not reduce development of embryos produced from Brahman oocytes (BL = 42.1 +/- 4.8% vs. 55.6 +/- 4.8% for 38.5 and 41 degrees C, respectively; ABL = 17.6 +/- 4.2% vs. 32.4 +/- 4.2%). In the second experiment, oocytes from Holstein cows were fertilized with semen from bulls of either Brahman or Angus breeds. Heat shock of embryos >or= 9 cells reduced development to BL (P < 0.002) and ABL (P < 0.005) for embryos sired by both Brahman (BL = 54.3 +/- 7.7% vs. 23.4 +/- 7.7%; ABL = 43. +/- 7.4% vs. 7.9 +/- 7.4%, for 38.5 and 41 degrees C, respectively) and Angus bulls (BL = 57.9 +/- 7.7% vs. 31.0 +/- 7.7%; ABL = 33.6 +/- 7.4% vs. 18.4 +/- 7.4%, for 38.5 and 41 degrees C, respectively). There were no breed x temperature interactions. Results suggest that the oocyte plays a more significant role in the resistance of Brahman embryos to the deleterious effects of heat shock than the spermatozoa.     

Factors affecting survivability of transferred whole and demi-embryos in a commercial dairy herd

Sixty Holstein donor cows were superovulated and embryos were collected during a 6-d (27 cows) and a 4-d (33 cows) period approximately 60 d apart. Forty-three donor cows yielded embryos. Ninety-one embryos graded 1 or 2 were split and transferred to 181 recipient Holsteins. Demi-embryos were graded 2, 2-, 3 and 3- prior to transfer. Pregnancy and calving percentages were similar for all demi-embryo grades, averaging 59 and 53% from the two donor groups, respectively. Twin demi-embryo pregnancies averaged 36 and 19% for embryos split at the compacted morula and blastocyst stages, respectively. Twin demi-embryo calvings averaged 30 and 15% for these same groups. Progesterone levels of recipients (of either whole or demi-embryos) of second period donors were measured. Pregnancy rate increased generally with level of progesterone; however, calving percentage was slightly greater for recipients with intermediate levels of progesterone (2-6 ng/ml). Multiparous cow (20) recipients of demi-embryos had 45% pregnancy and 40% calving, while nulliparous heifer (161) recipients averaged 59 and 53% pregnancy and calving, respectively.     

Dose of FSH-P as a source of variation in embryo production from superovulated cows

Various superovulation treatments were evaluated retrospectively in a commercial embryo transfer program. When it appeared that embryo production was dependent on the dose of FSH-P, a dose response curve to FSH-P was developed and embryo production compared using several treatment regimes. There was a significant effect of dose of FSH-P on embryo production in superovulated cows. At doses in excess of 28 mg, embryo production declined from 5.9 transferable embryos per collection (28 mg) to 2.7 (60 mg). Total embryos collected declined from 14.9 to 6.8 and the percent transferable from 57% to 40%. There was no advantage in using a five-day treatment over a four-day treatment regimen or in using a level over a declining dose regimen. There was a large individual variation in cow response rendering decisions on treatment changes based on single records unreliable. The percentage of zero collections increased with dose rate. Adoption of a 28-mg dose rate in commercial donors resulted in the embryo production forecast by these studies.     

Embryo production and quality of Holstein heifers and cows supplemented with beta-carotene and tocopherol

The hypothesis was that the intramuscular injection (i.m.) of beta-carotene associated to tocopherol improves cow (n=86) and heifer (n=91) embryo production and quality. Time of estrus was synchronized in animals with an ear implant with 3 mg of norgestomet associated with an i.m. injection of 6 mg of norgestomet and 10mg of estradiol valerate (CRESTAR, Intervert International B.V., Boxmeer, Holland) and superovulated by 8 i.m. FSH/LHp injections (400 IU-heifers and 500 IU-cows) in decreasing concentrations at 12h intervals. Animals were inseminated 12 and 24h after observed onset of estrus and embryos recovered 7 days later. Animals were randomly allocated to one of three treatments: (1) vegetable oil vehicle (control), (2) 800 mg of beta-carotene and 500 mg of tocopherol (T800) and (3) 1200 mg of beta-carotene and 750 mg of tocopherol (T1200). Supplemental injections were given at the day norgestomet implants were inserted and at first superovulatory injection. An index (Embryo Quality Index or EQI) was proposed to more precisely evaluate embryo quality (excellent*1 + good*2 + regular*3 + poor*4 + degenerate*5 + unfertilized ova*5)/total. There was an interaction between physiological stage (heifer or cow) and treatment on EQI (P=0.01) and on the proportion of viable embryos (P=0.03), where both variables were improved in T1200 cows, but not in heifers. The average EQI for heifers and cows in control, T800 and T1200 were 2.6+/-0.3 and 3.6+/-0.3; 2.5+/-0.3 and 3.6+/-0.3; 2.9+/-0.3 and 2.7+/-0.3, respectively. The average total number of viable embryos was greater (P=0.01) in supplemented cows (3.5+/-1.1; 5.4+/-1.4 and 7.5+/-1.2 in control, T800 and T1200, respectively), but less (P=0.01) in heifers (7.5+/-1.2; 5.6+/-1.2 and 4.0+/-1.1 in control, T800 and T1200, respectively). Supplementation injections of beta-carotene associated to tocopherol improved embryo quality in superovulated Holstein cows, in the present experimental conditions and may be advantageous in similar embryo production systems. However, at dosages applied in the present experiment, this treatment should not be recommended for nulliparous heifers.     

The use of embryo transfer to produce pregnancies in repeat-breeding dairy cattle

Repeat breeding is an important factor affecting economic success in dairy management. The objective of this study was to investigate the effectiveness of transfer of frozen-thawed IVF embryos in establishing pregnancy in repeat-breeding Holstein cattle. Cumulus oocyte complexes were collected by aspiration of 2-5 mm follicles from ovaries obtained at two local abattoirs. After IVF, days 7 and 8 blastocysts were frozen either in 1.5M ethylene glycol with 0.1M sucrose, or in 1.4M glycerol with 0.1M sucrose. Holstein recipients (122 heifers and 410 cows) included those that had not conceived after 3-21 inseminations. Embryos frozen in ethylene glycol were transferred directly, and embryos frozen in glycerol were transferred after dilution of the cryoprotectant in sucrose into recipients 7 or 8 days after estrus (without-AI group), or following AI (with-AI group). Pregnancy rates were compared by the Chi-square test. Significantly higher pregnancy rates were achieved by embryo transfer following AI (with-AI group) than by embryo transfer alone (without-AI group) in both heifers (49.2 and 29.5%, respectively) and cows (41.5 and 20.4%, respectively; P<0.05). Pregnancy rates were not significantly different between heifers and cows. However, pregnancy rate decreased as the number of inseminations prior to embryo transfer increased in the with-AI group, but not in the without-AI group. Therefore, transfer of frozen-thawed IVF embryos during the same cycle in which AI was done improved pregnancy rates in repeat-breeding Holstein heifers and cows, and suggested that embryo transfer is an alternative in the treatment of repeat breeding.     

Interspecific transfer of IVM IVF-derived red sheep (Ovis orientalis gmelini ) embryos to domestic sheep (Ovis aries )

Two embryo production methodologies were investigated to generate Red sheep embryos for use in an interspecific embryo transfer program. In Experiment 1, 4 multiparous female Red sheep (Ovis orientalis gmelini ) were implanted with CIDR type G devices for 11 d. Forty-eight hours prior to CIDR removal, a total of 22.5 mg bid of FSH-P was administered over a 3-d period. Laparoscopic embryo collection was performed 5 d post breeding, and embryos were transferred to domestic recipient ewes (Ovis aries and Ovis orientalis musimon ). In Experiment 2, 7 nulliparous female Red sheep were implanted with CIDR devices and injected with 200 IU of PMSG and 25 mg of FSH-P on the 8th day of implant insertion. At 60 to 70 h post PMSG/FSH-P treatment, follicular oocytes were aspirated laparoscopically. The recovered oocytes were matured in M199 (with fetal calf serum, FSH, LH, penicillin and streptomycin) at 39 degrees C in a humidified atmosphere containing 5% CO(2). At 24 h oocytes were fertilized with frozen-thawed semen at a concentration of 1.6 x 10(6) sperm/ml. The ova/embryos were placed in CR2 or BOEC culture medium at 20-22 h post IVF. Following 3 to 4 d in culture, embryos were transferred laparoscopically to the uterine horn of synchronized recipients. In Experiment 1, 4 embryos and 6 UFO were collected from 2 embryo donors, respectively. Two embryos were transferred with the aid of a laparoscope to each of 2 Rambouillet recipients, one of which gave birth to a healthy Red sheep lamb at 158 d of gestation. In Experiment2, a total of 62 oocytes was collected from 7 oocyte donors; 16 developed to the 16- to 32-cell stage and were transferred to 8 recipients. Three of these IVM-IVF embryos were transferred laparoscopically to 2 Mouflon recipients, resulting in no pregnancies. Thirteen IVM-IVF embryos were transferred to 6 Rambouillet recipients. Each of these gave birth to a single healthy Red sheep lamb. Gestation lengths of the 3 IVM-IVF lambs ranged from 152 to 162 d. This research demonstrates that when using compatible species IVM-IVF technology in conjunction with interspecific ET can lead to the production of live offspring and can be used to propagate exotic ovine species.     

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.