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I. Wilmut, D. I. Sales and C. J. Ashworth

Introduction

Substantial prenatal mortality has been observed in all mammals studied, although there are significant differences between species in the extent and timing of the death. There are two reasons why it is important to define the causes of such loss. First, it is of fundamental interest to understand why prenatal loss continues to occur despite natural selection for efficient reproduction. Second, there may be practical applications arising from such knowledge if it can be used to increase the survival of embryos. Great distress is caused to those people who suffer infertility, particularly if it is a result of repeated miscarriage. Furthermore, a substantial economic loss follows prenatal death in farm animals. This death leads to a reduction in litter size in pigs and prolific sheep and, in cattle and sheep with only one ovulation at each oestrus, an increased interval between births.

The causes of prenatal mortality have been

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C. J. Ashworth, D. I. Sales and I. Wilmut

Summary. Plasma progesterone concentration and embryo survival were determined during successive pregnancies in ewes throughout one breeding season. The probability of an embryo surviving was associated with the progesterone concentration on the days around ovulation, with the timing of the increase from periovulatory to luteal values, and with the rate at which progesterone concentrations increased. Individual embryo survival decreased both as the number of corpora lutea increased, and towards the end of the breeding season; the latter effect could be explained entirely by differences in progesterone concentration. Considerable variation in progesterone secretion and in embryo survival was observed within the same ewes during successive pregnancies. Such variability in progesterone concentrations during early pregnancy may be a cause of some embryo mortality.

Keywords: embryo survival; fertility; progesterone; sheep; season

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C. Biggs, J. E. Tilton, J. Craigon, G. R. Foxcroft, C. J. Ashworth and M. G. Hunter

Comparisons were made between characteristics of pre-ovulatory follicles recovered from prolific Chinese Meishan gilts (n = 12) and from European Large-White hybrid gilts (n = 13) in the late follicular phase preceding their fifth oestrous cycle, to determine whether there is an ovarian basis for the enhanced prolificacy in the Meishan. A total of 177 follicles per breed was classified as pre-ovulatory, based on follicular fluid oestradiol concentrations. Results obtained demonstrated high variability in all follicular characteristics in both breeds and no decrease in heterogeneity was evident in the Meishan. The Meishan follicles tended to be smaller (P < 0.06) and had less follicular fluid (P < 0.005), but total oestradiol content per follicle was similar (P > 0.1) with the result that the concentration of oestradiol in follicular fluid tended to be higher (P < 0.06) in Meishan than Large-White hybrid pigs. There were no differences between breeds in terms of testosterone concentration in follicular fluid, hCG binding to granulosa cells or total DNA content of granulosa cells. Concentrations of inhibin in follicular fluid were similar in both breeds (P > 0.1) which resulted in a trend towards less total inhibin content in Meishan than Large-White hybrid follicles (P = 0.065). Corpora lutea were recovered from both breeds (n = 12 per breed) on days 27–31 of pregnancy after mating at first, second and third oestrus:corpora lutea were smaller (P < 0.001) and contained less progesterone per corpus luteum in the Meishan (P < 0.05) than in Large White hybrid pigs. However, since ovulation rate was higher in these particular Meishan pigs (P < 0.005), total ovarian progesterone content per animal was similar in both breeds. These results demonstrate that there was no decrease in the variability in follicular characteristics from Meishan pigs. However, both follicles and corpora lutea were smaller in Meishan than in Large-White hybrid pigs, and progesterone per corpora lutea was also lower in the Meishan. In spite of their smaller size, Meishan follicular oestradiol content was similar to that of Large-White hybrid pigs, such that the oestradiol concentration in follicular fluid tended to be higher in Meishan follicles.

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C. J. Ashworth, C. S. Haley, R. P. Aitken and I. Wilmut

Summary. Embryos were transferred between Meishan and Landrace × Large White (control) gilts on Day 4 or 5 to establish approximately equal numbers of all four possible combinations of donor breed and recipient breed. The breed of the donor gilt significantly (P < 0·01) affected embryo survival with 44·5% of transferred Meishan embryos and 69·6% of transferred control embryos surviving to Day 30 ± 1. There was no influence of the breed of the recipient gilt on the proportion of embryos which survived. These differences in embryo survival between the two breeds could not be explained by differences in (1) the number of embryos transferred, (2) the stage of development of the embryos transferred, (3) the interval between ovulation and transfer or (4) the degree of asynchrony between donor and recipient gilt.

On Day 30 ± 1 embryos from control donors developed into longer fetuses (P < 0·01) with larger allantoic sacs (P < 0·05) than did embryos from Meishan donors. Fetuses in control recipients were longer (P < 0·01), heavier (P < 0·001) and had larger allantoic sacs (P < 0·05) than fetuses occupying Meishan uteri. The interaction between breed of donor gilt and breed of recipient gilt did not significantly affect conceptus growth.

These results suggest that Meishan pig embryos may be less tolerant to routine embryo transfer procedures than those of control gilts, that the genotype of the dam does not affect the proportion of embryos surviving to Day 30 ± 1. and that both fetal and maternal factors affect conceptus growth.

Keywords: Meishan pig; embryo transfer; embryo survival; conceptus growth

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I. Wilmut, W. A. Ritchie, C. S. Haley, C. J. Ashworth and R. P. Aitken

Summary. A comparison was made of the rate and uniformity of development of embryos recovered from Meishan and European white sows. The time of ovulation was estimated to be 34·3 and 49·0 h after the onset of oestrus in large white and Meishan sows, respectively. Embryos were recovered from a total of 38 Meishan and 37 European pigs between 18 and 219 h after the estimated time of ovulation. Embryos recovered after 18–59 or 44–82 h were classified into one of 11 stages (from early fertilization to early blastocyst), and the maximum blastocyst diameter was measured for embryos recovered 140–219 h after ovulation. There was no evidence of a difference between the genotypes in the stage or size of embryos at these times or of large differences between the genotypes in the extent of variation in embryo stage within females, although a minority of European white females had very variable embryos. As the differences between the embryos of the Meishan and the European white were small, it seems unlikely that greater uniformity of Meishan embryo development is a major cause of the higher prenatal survival in that breed.

Keywords: pigs; prenatal survival; embryo development

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I. Wilmut, C. J. Ashworth, A. J. Springbett and D. I. Sales

Summary. Embryos at different stages of development were transferred to recipient ewes on Day 6 to investigate the effect of variation in stage of development on embryo survival and growth. Three groups of ewes received 2 embryos that were at the same stage of development, Day 4, Day 6 or Day 8. A fourth group received 1 Day-4 and 1 Day-8 embryo. At autopsy on recipient Day 34 there were no significant differences in embryo survival (Day 4, 34%; Day 6, 50%; Day 8, 46%; and Day 4 and 8, 48%). Fetuses developing from Day-8 embryos were heavier than others (Day 4, 1·10 ± 0·06 g; Day 6, 1·15 ± 0·06 g; Day 8, 1·41 ± 0·08 g; P < 0·05). In Group 4 neither survival nor growth of embryos was significantly affected by the presence of an embryo at a different stage of development.

The ability of the uterus to stimulate development of a relatively retarded embryo is confirmed. Apparently the uterus has less effect in slowing the development of advanced embryos.

Keywords: embryo transfer; embryo survival; embryo growth; sheep

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E M Ferguson, J Slevin, M G Hunter, S A Edwards and C J Ashworth

The present study examined the effects of feeding gilts a high fibre diet from the third post-pubertal oestrus until either day 19 of the same cycle or insemination at the following oestrus on oocyte maturity, embryo survival and associated changes in reproductive hormone concentrations. Gilts fed with the high fibre diet had lower circulating oestradiol concentrations on days 17, 18 and 19 of the cycle and increased LH pulse frequency on day 18. More oocytes recovered on day 19 from gilts receiving the high fibre diet were at metaphase II after 46-h culture in medium containing 10% of their own follicular fluid, despite fewer large (>7 mm) follicles in these gilts when compared with control animals. There was no effect of diet on ovulation rate, corpora lutea size or progesterone concentrations on days 10–12 after insemination, but embryo survival on days 27–29 after insemination was higher in gilts that received the high fibre diet. This study demonstrates that a high fibre diet that increases embryo survival also improves oocyte maturity and provides information on endocrine correlates that may shed light on underlying mechanisms.

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H N Jones, C J Ashworth, K R Page and H J McArdle

Trans-placental transport of amino acids is vital for the developing fetus. Using the BeWo cell line as a placental model, we investigated the effect of restricting amino acid availability on amino acid transport system type A. BeWo cells were cultured either in amino acid-depleted (without non-essential amino acids) or control media for 1, 3, 5 or 6 h. System A function was analysed using α(methyl-amino)isobutyric acid (MeAIB) transcellular transport studies. Transporter (sodium coupled neutral amino acid transporter (SNAT1/2)) expression was analysed at mRNA and protein level by Northern and Western blotting respectively. Localisation was carried out using immunocytochemistry. MeAIB transcellular transport was significantly (P < 0.05) increased by incubation of the cells in amino acid-depleted medium for 1 h, and longer incubation times caused further increases in the rate of transfer. However, the initial response was not accompanied by an increase in SNAT2 mRNA; this occurred only after 3 h and further increased for the rest of the 6-h incubation. Similarly, it took several hours for a significant increase in SNAT2 protein expression. In contrast, relocalisation of existing SNAT2 transporters occurred within 30 min of amino acid restriction and continued throughout the 6-h incubation. When the cells were incubated in medium with even lower amino acid levels (without non-essential plus 0.5 × essential amino acids), SNAT2 mRNA levels showed further significant (P < 0.0001) up-regulation. However, incubation of cells in depleted medium for 6 h caused a significant (P = 0.014) decrease in the expression of SNAT1 mRNA. System L type amino acid transporter 2 (LAT2) expression was not changed by amino acid restriction, indicating that the responses seen in the system A transporters were not a general cell response. These data have shown that placental cells adapt in vitro to nutritional stress and have identified the physiological, biochemical and genomic mechanisms involved.

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S C Fernando, J S Buck, M D Ashworth, J W Ross, R D Geisert and U DeSilva

Previous studies have suggested that the porcine endometrium may express several tissue kallikreins during the estrous cycle and early pregnancy. The present study investigated porcine endometrial and conceptus tissue kallikrein 1, 4, 11, and 14 mRNA expression during the estrous cycle and early pregnancy. Tissue kallikrein (KLK) gene expression was evaluated using quantitative RT-PCR and in situ hybridization. KLK1 expression was similar across the estrous cycle and early pregnancy, and localized to the endometrial luminal (L) and glandular (G) epithelium. KLK4 endometrial mRNA expression was greatest on days 0, 5, and 10 when compared with days 12, 15, and 17 of the estrous cycle and greater in cyclic compared with pregnant gilts. Expression of KLK4 was more intense in the stroma and uterine epithelium from days 0 to 10 of the estrous cycle. Endometrial KLK11 mRNA was not different between cyclic and pregnant gilts but the expression was greatest on days 10 and 12 compared with all other days evaluated. There was an increased intensity of KLK11 gene expression in the stratum compactum on day 10 of the estrous cycle and early pregnancy. Endometrial KLK14 mRNA expression was not detectable on days 5 and 10 but was expressed on days 0, 12, 15, and 17 of the estrous cycle and pregnancy. KLK14 expression was localized in the uterine L and G epithelium, and stroma throughout the endometrium after day 10. Conceptus KLK1 mRNA did not change from days 10 to 17 of gestation. However, conceptus KLK4, and 14 mRNA expression was greatest on day 10 with expression declining after day 14 of gestation. Expression of the various tissue kallikreins in the endometrium and conceptus during the estrous cycle and early pregnancy in the pig can serve in the activation of growth factors and tissue remodeling during the establishment of pregnancy.