Effect of asynchronous transfer on bovine embryonic development and relationship with early cycle uterine proteome profiles.

The uterus provides the nurturing environment that supports the growth of the early preimplantation bovine conceptus. To determine critical time points of uterine influence, in vitro-produced Day 7 blastocysts were transferred into synchronous (Day 7) uteri and asynchronous uteri (Days 5 or 9). Embryo growth was evaluated 7 and 15 days after transfer and compared with that of embryos generated by AI. Conceptuses recovered from asynchronous Day 9 transfers were fourfold larger than synchronous transfer or gestational Day 14 AI conceptuses; by 15 days after transfer, differences were less marked. Two-dimensional gel electrophoresis was used to compare the histotroph protein composition of uterine luminal flushings (ULF) on Days 5 and 9 after oestrous to determine any protein differences that would promote embryo growth. The ULF were collected by serially flushing the uteri of the same heifers and mature cows at different times of the cycle. Ten proteins that differed in abundance between Day 5 and 9 were identified by mass spectrometry. Three, namely phosphoserine aminotransferase 1, purine nucleoside phosphorylase and aldose reductase, were verified by western blot analysis as more abundant on Day 9 (P<0.002). Myostatin was present in only in Day 9 ULF, whereas tissue inhibitor of matrix metalloproteinase 2 (TIMP2) and legumain were only detected in Day 14 ULF. Although mature cows had lower progesterone concentrations on Days 5 and 14 (P<0.05) and tended to have less TIMP2 than heifer groups, no other protein differences were detected. Thus, the embryo growth-enhancing environment on Day 9 was associated with temporal changes in the expression of several proteins of the histotroph.

Microencapsulation of bovine spermatozoa for use in artificial insemination: a review.

A technique for microencapsulation of bovine spermatozoa has been developed with minimal spermatozoal injury and thus of potential use in artificial insemination. The polymers poly-l-lysine, polyvinylamine and protamine sulfate have proven best for membranes. Encapsulation has been successful with capsules ranging in size from 0.75 to 1.5 mm, and with sperm concentrations from 45 to 180 x 10(6) cells mL-1. Successful extenders include CUE, CAPROGEN, and egg yolk-citrate-glycerol (maximum 10% v/v egg yolk for normal capsular shape). Capsule fragility (ability to rupture under ageing and physical stress) is negatively related to membrane thickness which ranges from 1.92 to 5.32 microns (depending on the concentration of polymer used) and positively related to concentration of sperm encapsulated. Heterospermic studies have shown that encapsulated sperm are capable of fertilization in vivo, but are at a disadvantage to unencapsulated sperm when cows are inseminated at conventional times. Uterine retention of inseminates is favoured by capsules having a 'sticky' membrane. Using current procedures, preliminary homospermic fertility studies indicate that sperm encapsulated with poly-l-lysine or protamine sulfate may achieve normal fertility.

Understanding maternal contributions to fertility in recipient cattle: development of herds with contrasting pregnancy rates.

Causes of variation amongst recipients within a herd in their ability to initiate and maintain pregnancy is largely unknown. In order to develop an experimental resource to understand the biology of recipient reproductive performance, each of 155 contemporary yearling heifers received 2 in vitro-produced embryos on 6 separate occasions during a 26-month period. Sixty days after transfer, pregnancy and the number of foetuses were determined ultrasonically and then pregnancies were terminated and the process was repeated. Heifers were ranked on their aggregate pregnancy rate performance, and the highest (High) and lowest (Low) 25 were retained. Mean pregnancy rates of all recipients ranged from 0.20 to 0.67 depending on transfer occasion. The mean +/- s.e. pregnancy rate of the High and Low sub-herds were 0.76+/-0.04 vs. 0.11+/-0.03, respectively (P<0.001), with 55% and 37% of this difference due to differences in Day 25 return to oestrus rates and losses between Day 25 and Day 35, respectively. We suggest that failure in the mechanism involved in maternal recognition of pregnancy was a major cause of the difference between the two sub-herds. These sub-herds are a unique experimental resource for understanding the early pregnancy process in cattle.

Statistical models predicting embryo survival to term in cattle after embryo transfer.

Embryo survival to term in recipient cattle is highly variable. We examined calving data in the published literature to determine whether a model of binomial independence or a model which includes an embryo (e) and recipient term (r), adequately explain observed embryo survival rates following attempts to induce twin calving using transfer of two embryos. To achieve this we examined 32 published papers which provided us with 47 sets of data concerning 4560 recipients with either 0, 1 or 2 calves born. In each set of data, the observed embryo survival rate to term (p) (number of calves born/number of embryos) was calculated and the expected number of recipients with either 0, 1 or 2 calves born was determined, assuming a binomial distribution. Parameters for the second model were estimated using maximum-likelihood procedures. The model of embryo independence was rejected in 85% of the sets of data, suggesting that factors other than the embryo are important sources of variation in embryo survival or loss. The proposed e and r model of embryo survival adequately describes the published data in recipients receiving either single or twin embryos. In general, only 50-70% of embryos and recipients are sufficiently competent to result in a calving. Variation among laboratories producing either in vitro or in vivo derived embryos was due to variation in recipient and not embryo competence. It is argued that e rather than observed embryo survival rate, and r rather than observed pregnancy rate, should be used to compare differences among embryo treatments and groups of recipients, respectively. Acceptance of this proposition should permit faster progress in identifying the biology of superior embryos and recipients, which is a prerequisite to improving embryo survival rate in cattle. Collectively, the published data are not consistent with a model of embryo independence, and that a model of embryo survival to term which recognises recipient as well as embryo contributions to embryo survival may be more appropriate in cattle.

Laparoscopic transfer of ovine and cervine embryos using the transpic technique.

A transpic technique was developed to transfer embryos to 352 sheep and 4 deer recipients using a laparoscope, a modified pair of Allis forceps and a modified Cassou aspic normally used for laparoscopic uterine insemination. The overall proportion of uncomplicated transfers in Experiment 1 in 216 recipient ewes was 90.7% (range between groups 80 to 100%), 3.7% of the transfers were presumed to be loss of embryos during expulsion from the transpic, and 5.6% were apparent transfers into the uterine wall. In Experiment 2,83% of transfers into 136 ewe recipients were uncomplicated, 5% were presumed to be loss of embryos during expulsion, 1% was apparent transfer into the uterine wall, and 11% involved 2 attempts at transfer. Only 34% of 116 recipients receiving low-quality frozen-thawed embryos were pregnant and 24% of the 226 embryos survived to term. In contrast, high pregnancy rates (>80%) and embryo survival rates (>70%) were achieved following uncomplicated and twice attempted transfers of fresh embryos. Pregnancy rates and embryo survival rates were low (<2%) following the presumed loss of embryos during expulsion and apparent transfers into the uterine wall. All 4 deer transfers were uncomplicated and 2 2 good-quality embryos survived to term compared with 0 2 low-medium quality embryos. The transpic technique is a moderately invasive technique which permits fast (15 to 20/h) and reliable transfer of embryos in small ruminants. With appropriate care, nearly all of the embryos can be correctly placed in the uterus, and high pregnancy rates and embryo survival rates can be achieved using this technique.

Transuterine embryo migration in recipient cattle.

Transuterine migration of bovine embryos following fertilization in vivo is apparently rare, but little is known about migration following embryo transfer. We studied heifers receiving either 1 or 2 in vitro produced embryos to determine 1) the incidence of transuterine migration, 2) the timing of migration and 3) the random or systematic occurrence of the event. In 4 experiments, 436 heifers received embryos and 218 of these were pregnant at necroscopy on either Day 14, Day 18, Day 26 or Day 60 of pregnancy. Overall, 43/218 (20%) of the heifers had embryos that had migrated. The frequency of migration was higher in twin (30/68) than in single (13/150) embryo transfers of pregnant recipients (44 vs 9%; P<0.001), and in contralateral (9/15) than in ipsilateral (33/170) transfers (60 vs 19%; P<0.001). Among the heifers that received embryos by ipsilateral transfer, the migration rate was similar to that in heifers pregnant with a singleton after the transfer of either 1 (2/48) or 2 (4/60) embryos (4 vs 7%, NS). The migration rate was highest at Day 26 (12/37) in heifers receiving twin embryos by ipsilateral transfer but was similar at all other stages of pregnancy (15/111, 32 vs 14%; P<0.01). Migration was first observed by Day 14, and it appears that either further migration occurred over the next 12 d or that migration was associated with a higher survival rate from Day 14 to Day 26. The low migration rate evident at Day 60 suggests that migration by Day 26 was associated with increased embryo or fetal death by Day 60. The data suggest that embryo migration is probably independent for each of a pair of surviving embryos. We conclude that in cattle embryo migration is embryo-dependent, but this capability is dormant unless more than 1 embryo is present in a uterine horn or the embryos are transferred to the contralateral uterine horn. The relationship between migration and embryo survival remains unclear.

Selected times of insemination with microencapsulated bovine spermatozoa affect pregnancy rates of synchronized heifers.

This experiment was designed to test whether spermatozoa encapsulated in an alginate poly-L-lysine matrix had an extended fertile life in vivo after insemination. Estrus was synchronized in 417 primiparous Friesian and Jersey heifers with a system based on a CIDR-B intravaginal device before the heifers were inseminated either during proestrus (24 h after device removal) or at estrus (48 h after device removal). Pregnancy rates to first inseminations did not differ between the 24 and 48 h inseminations (61 vs 60.6%) with liquid semen diluted in Caprogen (control) but differed with encapsulated semen (45.1 vs 68.6%). The difference in pregnancy rates between the 2 types of semen was more pronounced (P < 0.08) in the animals that were visually detected in estrus. The mean survival time of spermatozoa in the female reproductive tract following insemination at the 24-h insemination time was estimated to be 50 +/- 7.5 h. The increased pregnancy rate with insemination of encapsulated spermatozoa at 48 h could have been due to this process predisposing spermatozoa to capacitate soon after insemination.

Embryo transfer in fallow deer (Dama dama): superovulation, embryo recovery and laparoscopic transfer of fresh and cryopreserved embryos.

Multiple ovulation-embryo transfer (MOET) protocols for farmed fallow deer (Dama dama) were investigated in a series of 3 experiments. A total of 37 donors, of either European (D.d. dama ; n = 30) or Mesopotamian hybrid (D.d. mesopotamica x D.d. dama ; n =7) genotype, each received an intravaginal silastic device containing 0.3 g progesterone (CIDR-type G device) for 14 d and injections of 0.5 units ovine FSH (8 x 0.06 unit injections from Days 10 to 14 of device insertion) and 100 IU PMSG (either with the first or last FSH injection). All donors received laparoscopic intrauterine inseminations of fresh semen (50 x 10(6) spermatozoa) from a Mesopotamian sire 36 h after withdrawal of CIDR devices. Embryos were recovered by laparotomy on Day 6 (Day 0 = estrus). Mean ovulation rates for the 3 experiments were 8.1, 9.8 and 7.0, with no effect of PMSG timing (P>0.10). However, embryo recovery rates, albeit low throughout the study (29.6%), were significantly improved with later PMSG administration (33.9 vs 20.1%; P<0.05). Hybrid and European donors performed in a similar manner. A range of embryo development stages was recovered throughout the study. In 2 experiments laparoscopic transfer of embryos to 48 recipient does treated previously with intravaginal CIDR devices for 14 d yielded a total pregnancy rate of 37.5%. In the experiment with fresh embryos, the use of clenbuterol to reduce uterine turgidity resulted in a higher proportion of does conceiving (3/4 ; 75%) compared with that of the untreated does (0/6 , 0%; P<0.05). In the second experiment, in which all the does routinely received clenbuterol, 10/19 (53%) and 5 19 (26%) does conceived following the transfer of fresh and cryopreserved embryos, respectively (P<0.05). While the overall efficiency of the MOET program was low (equivalent of 0.9 to 1.0 surrogate pregnancies per donor), improvements in the recovery rate of transferable embryos have considerable potential for genetic improvement of farm stock and captive propagation of endangered Mesopotamian fallow deer through maternal surrogacy programs.

Lamb birth weight is affected by culture system utilized during in vitro pre-elongation development of ovine embryos.

It has previously been reported that ovine embryos cultured in Synthetic Oviduct Fluid medium supplemented with 20% human serum (SOF+HS) develop into lambs with a high birth weight. We have investigated this phenomenon by culturing ovine zygotes in SOF+HS or a serum-free version of Synthetic Oviduct Fluid with BSA and amino acids (SOFaaBSA) in place of serum. Zygotes were either obtained from superovulated and naturally mated ewes or produced in vitro. Embryos were subsequently transferred to synchronized recipient ewes (n = 63). An additional group of ewes (n = 16) served as flock fertility and lambing controls. Development of zygotes to stages suitable for transfer (i.e., good to excellent compact morulae or blastocysts) was not affected by medium (SOFaaBSA = 53 +/- 5% vs. SOF+HS = 59 +/- 5%) but was affected by source (in vivo-derived = 74 +/- 5% vs. in vitro-derived = 35 +/- 5%, p < 0.001). Embryos incubated in SOF+HS were morphologically different from those incubated in SOFaaBSA, having abundant lipid droplets. Pregnancy rate (65%) and embryo survival (48%) of recipients determined by ultrasonography on approximately Day 60 of pregnancy did not differ between medium treatments or source of embryo. Mean weight of lambs from embryos cultured in SOF+HS (4.2 +/- 0.2 kg) was significantly heavier than that of controls (3.4 +/- 0.2 kg, p < 0.01) or of lambs from embryos cultured in SOFaaBSA (3.5 +/- 0.2 kg, p < 0.05). Furthermore, mean gestation length was longer in recipients receiving embryos incubated in SOF+HS (147 +/- 1 days) than in SOFaaBSA (145 +/- 1 day, p < 0.05). Reasons for this birth weight and gestation length difference are unclear, but our data suggest that different culture conditions can produce embryos with differing morphology, apparent chemical composition, and rate of development, resulting in lambs with differing gestation length and birth weight.