R.G. Watt

In vivo oocyte recovery and in vitro embryo production from bovine donors aspirated at different frequencies or following FSH treatment.

The effects of frequency of follicular aspiration and treatment of donor cattle with FSH on in vivo oocyte recovery and in vitro embryo production were studied. Simmental heifers (n = 24) formed 8 replicates of 3 treatments in which oocyte donors were aspirated 1) once a week, 2) twice a week, or 3) once a week following treatment with FSH for 3 d prior to aspiration. Oocytes were graded, washed, matured for 20 to 24 h and then inseminated with frozen/thawed semen from a single sire, followed by co-culture on granulosa cell layers. Embryo development was observed until Day 7 after insemination. Significantly fewer follicles per heifer per week were counted (14.7+/-2.3 vs. 27.4+/-3.1 vs. 23.1+/-2.8) and aspirated (12.0+/-2.0 vs. 21.8+/-2.7 vs. 20.1+/-2.6) in heifers on the once-weekly than twice-weekly aspiration treatment (P<0.01) or on the once-weekly aspiration after FSH treatment (P<0.05). There were no significant differences between treatments in the total number of oocytes recovered per week (5.6+/-1.2 vs. 8.9+/-1.5 vs. 6.1+/-1.2), but significantly more oocytes per heifer per week recovered from animals treated with FSH were graded Category 1 (2.8+/-0.4), i.e., >4 layers good cumulus with a clear, even cytoplasm, than from animals aspirated once (0.9+/-0.2; P<0.01) or twice a week (1.5+/-0.3; P<0.05). The number of transferable morulae plus blastocysts produced per heifer per week was higher from animals aspirated twice a week (2.4+/-0.4; P<0.05) or once a week following FSH treatment (2.1+/-0.4; P<0.05) than from animals aspirated once a week without FSH treatment (1.0+/-0.3). In conclusion, FSH treatment of bovine oocyte donors aspirated once a week enabled a similar number of transferable embryos to be produced per donor week as aspiration twice a week without FSH treatment. These 2 treatments produced twice as many transferable embryos per donor week as aspiration once a week without FSH treatment.

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.

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.

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.

Number of ovulations in texel x Scottish hill ewes carrying the inverdale fecundity (FecXI) gene

FECUNDITY genes affecting the number of ovulations have been identified in several sheep breeds worldwide (Davis 2004). The Inverdale fecundity gene was first reported in a Romney flock in New Zealand. This gene is located on the X chromosome, and is denoted FecXI. Sires carrying the gene will transmit it to all their female offspring, resulting in either heterozygous or homozygous carrier offspring, depending on the dam’s genotype. Increased prolificacy is found only in heterozygous carriers (Davis and others 1992a). Homozygous carriers are sterile due to abnormal ovarian development in the form of ‘streak ovaries’, which show no sign of follicular activity (Davis and others 1992b). Contrary to these effects of the gene on female carriers, no abnormal developmental effects of the FecXI gene have been reported in male carriers (McNatty and others 2003). Naturally occurring prolificacy genes have not been reported in Scottish hill breeds. As the husbandry systems in which breeds evolve may well influence whether prolificacy genes are retained (Davis and others 2006), it is improbable that harsh native hill environments would have favoured the establishment of such genes in these populations. This short communication describes an on-farm study to investigate the effect of the Inverdale gene on ovulation numbers in offspring when the gene was introduced, via a Texel carrier sire, into north country Cheviots and Scottish blackfaces farmed under favourable conditions. Numbers of ovulations were measured at the ewe lamb stage for Inverdale Texel and control Texel crossbred offspring from both dam breeds, and at the adult ewe stage for crossbred offspring from the Scottish blackface dams. Two flocks of hill ewes, one of 112 north country Cheviots and the other of 125 Scottish blackfaces, located on separate commercial farms in the north of Scotland, were artificially inseminated by laparoscopy using frozen semen from Texel rams that were either carriers or non-carriers of the FecXI gene. The offspring were born in late spring to early summer 2003 and reared commercially at pasture with their dams. The ovarian activity of the female offspring was recorded by laparoscopic viewing of the ovaries at the pubertal age of approximately seven months on one or two occasions. Animals with a detectable corpus luteum (CL) at first examination were not re-examined; those without a CL were re-examined nine or 10 days later. A blood sample was taken from animals with a detectable CL, centrifuged, and the plasma was assayed for progesterone. The animals’ liveweight and body condition score (BCS) on a five-point scale (Russel and others 1969) were recorded at the time of first ovarian examination. The ovarian activity of the offspring of the Scottish blackface flock was again recorded in autumn 2004, when they were approximately 18 months old. As before, animals without a CL at first examination were re-examined nine days later. Again, a blood sample was taken for a progesterone assay from those with a detectable CL. Liveweight and BCS were recorded at the first examination. For all laparoscopic procedures, animals were sedated by intramuscular injection of 0·6 ml 10 mg/ml acepromazine maleate BP (C-Vet). Local anaesthesia was given at trochar and cannula entry sites by subcutaneous injection of lignocaine hydrochloride BP/adrenaline BP (Locovetic; Bimeda). A single experienced operator performed all laparoscopies. Plasma progesterone concentrations were measured by radioimmunoassay in duplicate 100 μl aliquots as described by McNeilly and Fraser (1987). The interand intra-assay coefficients of variation were 15·3 and 12·7 per cent, respectively, and the minimum detection limit was 0·54 ng/ml. Liveweight and progesterone data were compared by analysis of variance, ovulation data and body condition scores by the Kruskal-Wallis test, and ovulation data (expressed as proportions) by chi-squared analysis. Data for the Texel x north country Cheviots and Texel x Scottish blackfaces are presented in Tables 1 and 2, respectively. None of the pubertal ewe lambs had more than two CLs. Among the north country Cheviot offspring that ovulated, carriers of FecXI had significantly more twin ovulations compared with the non-carrier controls. Of the 44 FecXI carriers that had ovulated, 16 (proportionally 0·36) had two CLs, compared with one of 16 (proportionally 0·063) for non-carrier flockmates (P<0·05). The presence of the Inverdale gene affected the incidence of twin ovulations in a similar manner in the offspring of the Scottish blackface flock. Of the 13 non-carriers that had ovulated, none had more than one CL, whereas there were 17 twin ovulations among the 40 FecXI gene carriers that had ovulated (P<0·05). Liveweight and BCS affected the number of ovulations in Texel x Scottish blackface FecXI carriers, with those having only one CL being significantly lighter and having significantly lower BCS (P<0·05) than those with twin ovulations. This effect was not observed in Texel x north country Cheviot carriers. Irrespective of genotype, animals with twin ovulations tended to have higher, albeit not statistically significant, progesterone concentrations than animals with single ovulations. Adult Texel x Scottish blackface carriers of the Inverdale gene had more ovulations than the controls (mean [se] 2·60 [0·08] v 1·85 [0·11]; P<0·01). The distributions of ovulations accounting for the increased mean are illustrated in Fig 1. Gene carriers that had more than two ovulations had significantly higher bodyweights than counterparts with one or two CLs, but there was no significant correlation between BCS and ovulation number (Table 3). FecXI gene carriers with Veterinary Record (2006) 159, 154-156