D.F. Dolman

Effect of frequency of follicle aspiration on oocyte yield and subsequent superovulatory response in cattle.

The effect of frequency of transvaginal follicular aspiration on oocyte yield and subsequent superovulatory response was studied in 2 experiments. In Experiment 1, 32 primiparous Hereford x Friesian cows were assigned to 4 treatments (n = 8 per treatment). Oocyte recovery was carried out once a week for 12, 8, 4 or 0 (control) wk. Embryo recovery for all animals was 7 wk after the completion of the aspiration schedules. In Experiment 2, the effects of oocyte recovery once or twice a week (n = 8 per treatment; control n = 18) for 12 wk and response to superovulation 4 wk after the last aspiration were compared using nulliparous purebred Simmental heifers. Increasing the period of once weekly aspirations from 4 to 12 wk (Experiment 1) did not affect the number of follicles observed per session (mean +/- SEM; 10.0 +/- 0.82) or aspirated (7.8 +/- 0.71), but the recovery rate of oocytes from follicles aspirated was greater for donors aspirated for either 4 or 8 wk than for 12 wk (32.3 +/- 3.73 vs 28.4 +/- 2.61 vs 20.1 +/- 2.13 %; P < 0.05). Following the last aspiration and prior to commencing superovulatory procedures, estrus or estrous activity was observed in 7 8 , 8 8 , 7 8 and 6 8 of the animals aspirated over 12, 8, 4 or 0 wk, respectively. Subsequent superovulatory responses and in vivo embryo recoveries were similar for all aspiration treatments and for control animals. Changing the frequency of oocyte recovery from once to twice weekly (Experiment 2) did not affect the numbers of follicles observed (9.1 +/- 0.63 vs 8.3 +/- 0.85), follicles aspirated (5.9 +/- 0.56 vs 6.2 +/- 0.69), oocytes recovered (1.7 +/- 0.27 vs 1.9 +/- 2.0) per session or the oocyte recovery rate (29.4 +/- 2.4 vs 30.4 +/- 2.4 %); nor was there any effect of frequency of aspiration on subsequent superovulatory response and embryo recovery. In conclusion, increasing the period of aspiration from 4 to 12 wk and the frequency from once to twice a week over 12 wk did not reduce the number of follicles observed or aspirated, or number of oocytes recovered per donor per session. Subsequent estrous cyclicity and responses to superovulation were unaffected by the periods or frequencies of oocyte recovery examined here.

Recipient management and embryo transfer

Abstract The recipient is one of the prime determinants of the success of the embryo transfer enterprise and improving recipient quality can produce significant savings in the cost of achieving a pregnancy. The effect of rejecting recipients or failure to achieve a pregnancy on the cost of a pregnancy was estimated by taking UK values of maiden recipients, interest charges, labour, feed, accommodation and drug costs. A period of 6 weeks from inclusion in the recipient pool to embryo transfer with pregnancy diagnosis occurring a further 7 weeks after transfer was assumed. The major changes in cost per pregnancy occur due to shifts in the proportion of those recipients synchronised which are detected in estrus; the proportion of those seen in estrus which are used for transfer; and the proportion of those used for transfer which become pregnant. Keeping two of these factors constant whilst changing the third gives estimates of their relative importance. The effect of an increase in the proportion of those synchronised which are detected in estrus from 85 to 95% is to give a saving of approx. 6% per pregnancy. A change in the proportion of those detected in estrus which are used for transfer from 80 to 90% produces a saving of approx. 7% per pregnancy. An improvament in the pregnancy rate of those used for transfer from 60 to 70% results in the greatest improvement in costs of approx. 14% per pregnancy. There are many factors involved in successful recipient management whose roles are closely related. The effects of genotype, parity, physiological status, health and stress are considered along with the nutritional, physical and climatic environment of the recipient. Further discussion centres on the synchronisation and detection of estrus, and the selection and management of recipients at transfer.

Embryo production using defined oocyte maturation and zygote culture media following repeated ovum pick-up (OPU) from FSH-stimulated Simmental heifers

To determine whether differences in ovarian follicle populations and endocrine status at ovum pick-up (OPU) influenced the quality and developmental competence of oocyte-cumulus complexes (OCCs) collected from follicle stimulating hormone (FSH)-stimulated donors, 24 Simmental heifers had their ovarian follicles aspirated via transvaginal ultrasound-guided OPU at both 15 (OPU1) and 21 (OPU2) days following a synchronised oestrus, on four consecutive occasions at 15-week intervals. More OCCs were collected during OPU1 than OPU2 (means +/- S.E.M. = 7.2 +/- 0.47 versus 5.7 +/- 0.44; P = 0.01), but the respective percentages that were of good quality (categories 1 and 2) did not differ significantly (55 +/- 3% versus 47 +/- 3%). The incidence of zygote cleavage following OCC maturation (Medium 199; protein-free), in vitro fertilization (mTALP; including 0.6% (w/v) albumin) and culture (modified SOF; protein-free) was not significantly different (mean +/- S.E.M. = 81 +/- 2% and 71 +/- 7% for OPU1 and OPU2, respectively). Corresponding blastocyst yields from good quality OCCs (24 +/- 3% and 26 +/- 4%) also did not differ. Although the same 3-day FSH regimen was used immediately prior to each OPU session, plasma FSH concentrations were consistently lower at OPU1 than OPU2 (1.3 +/- 0.28 ng/ml versus 2.5 +/- 0.45 ng/ml; P < 0.05). In contrast, plasma progesterone concentrations were higher at OPU1 (6.6 +/- 0.48 ng/ml versus 3.9 +/- 0.53 ng/ml; P < 0.001), with concentrations at OPU2 being consistent with the presence of luteal tissues, including both persistent corpora lutea and luteinised follicle remnants following OPU1. Failure of the significant differences in follicular and endocrine status between OPU1 and OPU2 to alter the developmental competence of OCCs suggests that, probably as a result of its stabilising influence on nutritionally-sensitive intraovarian regulators of oocyte competence, the constant feeding regimen had a more profound effect on oocyte quality than observed shifts in the peripheral concentrations of some reproductive hormones. Finally, the study demonstrates that it is possible to generate acceptable numbers of in vitro blastocyst-stage embryos from high genetic merit heifers using strategies which restrict reliance on protein to the in vitro fertilization stage of the production process.

Synchronization of estrus in embryo transfer recipients after using a combination of PRID or CIDR-B plus PGF2α

The use of CIDR-B or PRID in combination with prostaglandin F2alpha (PGF2alpha) for synchronizing estrus in embryo transfer recipients was evaluated in 2 experiments. In Experiment 1, virgin heifers (n=263) were synchronized using either a PRID (including estradiol benzoate capsule) or a CIDR-B in a combined program in which devices were inserted on Day 1, an injection of prostaglandin was given on Day 6, and devices were withdrawn on Day 7. The interval from device removal to the onset of estrus was significantly shorter for CIDR-B than for PRID-treated animals (50.44 vs 55.50 hours; P<0.003). The CIDR-B treatment resulted in a similar degree of synchrony to the PRID treatment (74.0 vs 70.4%; P=0.68). InExperiment 2, cows (n=95) and heifers (n=93) were allocated at random to be synchronized using a PRID (excluding estradiol benzoate capsule) plus PGF2alpha or a CIDR-B device plus PGF2alpha. The devices were inserted on Day 1, an injection of prostaglandin was given on Day 10 and the devices were removed on Day 12. Estrus was observed earlier following the CIDR-B treatment (43.50 vs 47.04 hours; P=0.01), but the degree of synchrony was similar (76.2 vs 76.3%; P>0.10) for the CIDR-B and PRID-treated animals. In both experiments, there were no significant differences in the proportions of animals observed in estrus, selected as embryo transfer recipients, or which achieved pregnancy consequent on embryo transfer between those synchronized using CIDR-B or PRID regimens. We conclude that the CIDR-B is a suitable device for synchronizing estrus in embryo transfer recipients.

Norgestomet implants, plasma progesterone concentrations and embryo transfer pregnancy rates in cattle

A study was undertaken to test the hypothesis that supplementation with exogenous progestagen at the time of embryo transfer would enhance pregnancy rates in recipients. Twohundred- and-seventy-two oestrus-synchronised crossbred heifer and cow recipients received 200 grade 1 and 72 grade 2 Simmental embryos transferred non-surgically. Heparinised blood samples were taken on day 6 and day 7 (oestrus = day 0) for the assessment of the endogenous plasma progesterone concentration. Half the recipients received an ear implant impregnated with 3 mg norgestomet on the day of embryo transfer. The pregnancy rates were 51.9 and 49.6 per cent for the norgestomet-treated and control groups, respectively. The pregnancy rate for grade 1 embryos was 56.0 per cent and for grade 2 embryos 36.1 per cent (P<0.01). The breed of recipient, weekday of transfer, operator and condition score had no effect on pregnancy rate. The maiden heifers had a higher pregnancy rate (54.2 per cent) than the cows (46.2 per cent). The mean plasma progesterone concentrations of the pregnant and non-pregnant groups on day 6 were 6.7 ng/ml and 6.6 ng/ml, respectively, and 7.6 ng/ml in both groups on day 7.

Effects of epidural injections and transvaginal aspiration of ovarian follicles in heifers used repeatedly for ultrasound-guided retrieval of ova and embryo production.

Postmortem examinations of 13 Simmental heifers that had received between 16 and 28 injections to induce caudal epidural anaesthesia, the last not less than seven months before they were slaughtered, showed that none of them had any evidence of infection or inflammation at the injection site or in adjacent bone and soft tissues. Seven of them had minor damage to intercoccygeal discs, consisting of discospondylosis with neovascularisation and chondroid metaplasia, consistent with injuries caused by needles. The severity of the damage was not related to the number of epidural injections received, suggesting that the damage was probably caused by a discrete suboptimal injection procedure. In a second study, the ovaries from 22 Simmental heifers that had undergone between 13 and 16 transvaginal follicular aspirations were examined postmortem. Approximately one-third of them had a natural texture with little or no evidence of scar tissue, and less than one in five had extensive scarring and a toughened texture. There was no evidence of compromised ovarian function, as determined by the number and normality of corpora lutea and large follicles, in any of the animals.

Follicular dynamics and superovulatory response in heifers

The study examined whether the response of heifers to exogenous gonadotrophin superovulatory treatment could be predicted from a knowledge of previous antral follicular dynamics. During a pretreatment monitoring phase, of 24 normal oestrous cycles (20.1 ± 0.33 days long) observed in 17 heifers, one, 15 and seven cycles showed one, two and three antral follicular waves respectively, as measured by ultrasonography. The subsequent ovulatory response (number of corpora lutea) to ovine FSH stimulation, after a CIDR-B/oestradiol benzoate/prostaglandin analogue cycle synchronisation regime, was not correlated with either oestrous cycle length or follicle wave number during the monitoring phase or with the number of follicles observed at the start of FSH treatment, but was related to the number of follicles observed during the monitoring phase (r = 0.47, P < 0.05). In conclusion, the present results show that the outcome of FSH superovulatory stimulation in heifers cannot be predicted from a knowledge of prior follicular dynamics.

Establishing twin pregnancies in cattle by embryo transfer

An experiment zoas conducted to assess differing methods of twin pregnancy establishment in Hereford × British Friesian beef cows and heifers. The experiment was 2 × 2 × 2 × 2 factorial design in which the factors were (i) source of embryos (in vivo or in vitro produced); (ii) pregnancy status of recipient (inseminated or non-inseminated); (Hi) method of embryo transfer (surgical or cervical); and (iv) uterine location of a native and transferred embryo, or two transferred embryos (both located in the ipsilateral, or one in each of the ipsi and contralateral uterine horns). Pregnancy and twinning rates for 285 animals used for embryo transfer were initially diagnosed at day 56 after induced oestrus by transrectal ultrasonography. Subsequently, calving rate and birth weiglit at calving were recorded. Pregnancy rates at day 56 after induced oestrus were similar for both surgical and cervical transfers (58·6% v. 55·2%), as was the case for twinning rate (36·2% v. 30·0%). Similarly, there were no differences between these two methods of transfer (50·0% v. 46·9%) and (26·1% v. 17·7%) for calving and twin calving rates respectively. Recipients which had two embryos located in the ipsilateral uterine horn had higher (P v. 47·3%) but similar twinning rates (32·6% v. 33·4%) at day 56 after induced oestrus to recipients which had one embryo located in each horn. A greater (P v. 41·0%) but fewer (P > 0·05) produced twins (17·8% v. 25·7%) than was the case for recipients which originally had one embryo located in each horn. In vivo produced embryos resulted in higher (P v. 39·7%) and twinning rates (48·3% v. 18·0%) at day 56, and higher (P v. 32·7%) and twin calving rates (36·3% v. 7·6%) than did in vitro produced embryos. Inseminated (Al + ET) recipients had slightly greater (P>0·05) pregnancy rates (61·6% v. 51·6%) and twinning rates (36·9% v. 28·7%) than non-pregnant recipients which received two embryos. A greater (P v. 42·0%) than was the case for non-pregnant recipients which received two embryos. The percentage producing twins at calving were similar for these two methods of twin pregnancy establishment. Embryo survival to day 56 after induced oestrus averaged 45·0% and was found to be non-independent of its co-twin. From day 56 to parturition foetal loss averaged 21·0% and foetal survival was found to be independent of the fate of its co-foetus. Twin foetuses located in the same uterine horn were lighter at birth than twin foetuses located in separate uterine horns (33·0 v. 35·2 kg; P

The effects of dose and pattern of administration of ovine FSH on the response to superovulation in performance tested, juvenile Simmental heifers.

The response of performance tested purebred Simmental heifers to various superovulatory treatments with ovine FSH was examined in two experiments. The heifers were 12 months old at embryo recovery, had average live weights of 468·4 kg (experiment 1) and 493·2 kg (experiment 2), and were fat (body condition score approx.4·0 to 4·5 units) at embryo recovery. In experiment 1, the effect of administering a total of 9·0 or 10·8 mg ovine FSH (Ovagen) administered as eight equal doses twice daily over 4 days was evaluated. In experiment 2, a total of 9·0 mg ovine FSH was administered either in equal doses in a level pattern or in declining doses twice daily over 4 days. The response to the low, compared with the high, dose of ovine FSH in experiment 1 was 8·8 v. 10·5 ovulations (corpora lutea); 7·7 v. 7·7 totalova plus embryos recovered; 6·0 v. 4·1 viable embryos; 4·4 v. 3·1 grade 1 embryos (P > 0·05); and 1·7 v. 4·1 non-fertile ova(F v. 10·7 ovulations; 5·6 v. 9·6 total ova plus embryos; 3·1 v. 5·6 viable embryos; 2·5 v. 3·8 grade 1 embryos; and 1·7 v. 2·2 non-fertile ova. It was concluded that, although the differences between the treatments in the yields of viable and grade 1 embryos were not significantly different, there are practical and economic advantages to using a low rather than a high level of gonadotropin and there are no marked disadvantages in these areas to administering ovine FSH in a declining rather than a level pattern of doses.