Fifty-one cyclic beef cows were mated with fertile bulls. At 36 h after the start of oestrus, cows were assigned to receive sesame oil (controls) or progesterone (100 mg) on Days 1, 2, 3 and 4 of pregnancy. Peripheral plasma concentration of progesterone was measured until slaughter on Days 5 or 14. Cows were randomly assigned to be slaughtered on Days 5 or 14 or remain intact and palpated per rectum on Day 40 to verify pregnancy. Uteri on Days 5 and 14 were flushed for recovery of luminal protein and conceptus tissue. Conceptus and endometrial tissues were cultured with [3H]leucine and submitted to two-dimensional-PAGE and fluorography. Administration of progesterone increased peripheral plasma progesterone concentration on Day 2-5. Conceptuses recovered from progesterone-treated cows on Day 14 were advanced in development compared to conceptuses from control cows. Conceptuses recovered from progesterone-treated cows were viable as polypeptides associated with maintenance of pregnancy in cattle were synthesized and released at an earlier time and pregnancy was maintained beyond Day 40. Early progesterone stimulation altered the synthesis and release of polypeptides from endometrial explant cultures on Day 5. Results indicate a role of progesterone in the maternal regulation of conceptus growth and development in early pregnancy of cattle.

/pdf/evidence-for-maternal-regulation-of-early-conceptus-growth-3nyhhsnamj.pdf

Evidence for maternal regulation of early conceptus growth and development in beef cattle

Milk samples were collected twice weekly from 535 dairy cows between parturition and the re-establishment of pregnancy to monitor ovarian activity by measurement of milk progesterone levels by radioimmunoassay. The mean progesterone levels after fertile and non-fertile first inseminations were similar from 21 days before until 13 days after insemination, those in non-pregnant animals declined while those in pregnant animals continued to rise until Day 22. Progesterone profiles of 47 repeat breeder cows (i.e. receiving greater than 3 inseminations) demonstrated that a variety of patterns was associated with the poor conception rate. A study of the post-partum interval showed that cows resumed cycles by 24 +/- 0.6 days after calving; the length of this interval varied significantly with the season of calving and non-significantly with the lactation number (i.e. age) of the cow, but was not related to yield. Of the 535 cows studied, 5.2% had not started to cycle within 50 days of calving and a further 5.2% resumed cycles which subsequently ceased. The progesterone profiles of acyclic cows indicated that after treatment with 0.5 mg LH-RH or a progesterone-releasing intravaginal device (PRID) for 14 days 75% of animals apparently ovulated but the calving to conception interval was not significantly altered compared with that of untreated control cows.

/pdf/milk-progesterone-levels-in-relation-to-conception-repeat-7ff44q5eht.pdf

Milk progesterone levels in relation to conception, repeat breeding and factors influencing acyclicity in dairy cows

The large offspring syndrome (LOS) is observed in bovine and ovine offspring following transfer of in vitro-produced (IVP) or cloned embryos and is characterized by a multitude of pathologic changes, of which extended gestation length and increased birthweight are predominant features. In the present study, we used bovine blastocysts to analyze cellular parameters, i.e., the number of cells in Day 7 blastocysts and the size of Day 12 elongating blastocysts, and molecular parameters, i.e., the relative abundance of developmentally important genes: glucose transporter (Glut) 1, Glut-2, Glut-3, Glut-4, heat shock protein (Hsp) 70.1, Cu/Zn-superoxide dismutase (SOD), histone H4.1, basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF) I receptor (R), and IGFII-R. Some blastocysts were produced by in vitro maturation and fertilization followed by in vitro culture in synthetic oviduct fluid medium supplemented with BSA or human serum or by in vivo culture in the sheep oviduct. Other blastocysts were derived in vivo from the uterine horns of superovulated donors. The findings made in the early embryos were related to a representative number of calves obtained from each production system and from artificial insemination (AI). In vitro culture of bovine embryos in the presence of high concentrations of serum or BSA significantly increased the number of cells in Day 7 blastocysts, the size of blastocysts on Day 12, and the relative abundance of the transcripts for Hsp70.1, Cu/Zn-SOD, Glut-3, Glut-4, bFGF, and IGFI-R when compared with embryos from the in vivo production groups. Birthweights of calves derived from IVP embryos were significantly higher than those of calves derived from sheep oviduct culture, superovulation, or AI. The results support the hypothesis that persistence of early deviations in development is causally involved in the incidence of LOS, in particular in increased birthweights. The cellular and molecular parameters analyzed in this study can be considered early markers of LOS in cattle.

https://bioone.org/journals/biology-of-reproduction/volume-67/issue-3/biolreprod.102.004481/Cellular-and-Molecular-Deviations-in-Bovine-In-Vitro-Produced-Embryos/10.1095/biolreprod.102.004481.pdf

Cellular and Molecular Deviations in Bovine In Vitro-Produced Embryos Are Related to the Large Offspring Syndrome

In the human, IVF and ET was developed for the treatment of tubal infertility, but it is already clear that the technique has wider applications. In this chapter, the present position of IVF and ET in infertility based on tubal, male factor and undiscovered causes (idiopathic) is discussed. There will be very rapid progress in the future, and thus some trends in research and extensions of IVF are also covered. It is anticipated that this procedure will greatly expand knowledge of the nature of human fertility disorders, particularly those that are currently obscure, such as male factor and idiopathic infertility.

Current Status and Future Prospects

Higher systemic progesterone in the immediate post-conception period is associated with an increase in embryonic growth rate, interferon-tau production and pregnancy rate in cattle. The objective of this study was to examine the effect of increasing progesterone concentration on Day 3 on subsequent embryo survival and development. Oestrus (Day 0) was synchronised in beef-cross heifers (n=210) and approximately two-thirds of the heifers were inseminated with semen from a proven sire, while the remainder were not inseminated. In order to produce animals with divergent progesterone concentrations, half of the animals received a progesterone-releasing intravaginal device (PRID) on Day 3 of the oestrous cycle, which was left in situ until slaughter. The four treatment groups were: (i) pregnant, high progesterone; (ii) pregnant, normal progesterone; (iii) non-pregnant, high progesterone; and (iv) non-pregnant, normal progesterone. Animals were blood-sampled twice daily from Days 0 to 8 and once daily thereafter until slaughter on Days 5, 7, 13 or 16, corresponding to the 16-cell stage, the blastocyst stage, the beginning of elongation and the day of maternal recognition of pregnancy, respectively. Embryos were recovered by flushing the tract with phosphate-buffered saline and characterised by stage of development and, in the case of Days 13 and 16, measured. Data were analysed by mixed models ANOVA, Chi-square analysis and Student's t-test where appropriate. Insertion of a PRID on Day 3 increased (P 0.05). While not significantly different, the proportion of viable embryos recovered was numerically greater in the high progesterone group on both Day 13 (58 v. 43%) and Day 16 (90 v. 50%). Elevation of progesterone significantly increased embryonic length on Day 13 (2.24+/-0.51 mm v. 1.15+/-0.16 mm, P=0.034) and Day 16 (14.06+/-1.18 cm v. 5.97+/-1.18 cm, P=0.012). In conclusion, insertion of a PRID on Day 3 of the oestrous cycle increased serum progesterone concentrations on subsequent days, which, while having no phenotypic effect on embryonic development on Days 5 or 7, was associated with an increase in embryonic size on Days 13 and 16.

Effect of increasing progesterone concentration from Day 3 of pregnancy on subsequent embryo survival and development in beef heifers

Of 226 donor cattle treated with PMSG to induce superovulation, 76.5% responded with 3 or more ovulations. Flushing at surgery or slaughter 10-16 days after oestrus recovered eggs and embryos that represented 49.3% of the number of ovulations. Of those recovered, 73.3% were embryos, an average yield of 4.0 embryos/treated cow or 4.8 embryo/flushed cow. The location of eggs and embryos was determined in 65 of the donors. Embryos and unfertilized eggs (6.1% of those recovered) were occasionally found in the oviducts. Empty zonae pellucidae were also found in the uterus on all days. The lengths, or diameters, of embryos were extremely variable within days and within donors, but mean values indicated logarithmic growth between Days 10 and 16. Eighty-four synchronous (+/- 1 day) recipients received single embryos, and 51 recipients twin embryos, by surgical transfer. Pregnancies were obtained in recipients up to Day 16 but not on Day 17, indicating the stage by which an embryo must be present to prevent luteolysis. The overall pregnancy rate at Day 42 was 50.4% and further 18.1% of the recipients exhibited extended oestrous cycles. Of 35 recipients that were allowed to go to term, 12 lost their pregnancies, most often between Days 42 and 63.

/pdf/collection-description-and-transfer-of-embryos-from-cattle-1d0b7jy111.pdf

Collection, description and transfer of embryos from cattle 10--16 days after oestrus.

Forty nine embryos, twenty unfertilized eggs and five other fresh eggs of 'doubtful' status have been recovered from 58 pony mares in 122 flushes up to 22 days after ovulation. The fresh egg or embryo recovery rate was 78% with surgical methods (or at slaughter) and 40-60% with non-surgical methods of recovery. The fertilization rate was about 70%. It has been confirmed that horse embryos normally enter the uterus as blastocysts 5-6 days after ovulation. Three features of early embryo morphology have become clearer upon comparison with unfertilized eggs of similar ages; early embryos are often ellipsoidal in shape; dispersal of most of a thick gel coat seems to be hastened by fertilization; gradual disappearance of refractile granules from the perivitelline space is similar in fertilized and unfertilized eggs. A tense, transparent, acellular capsule (considered to be different from the zona pellucida) is acquired by the spherical blastocysts within the uterus and persists at least until a diameter of 34 mm is attained (at 21 days in the present series). The capsule seems to be analogous, in part, with the 'neozona' described in rabbit blastocyst before attachment, and trophoblastic cells appear to be involved in its formation. Cleavage stages of oviductal embryos and diameters of uterine blastocysts from this series have been described and illustrated and used to extend previous knowledge of early growth patterns in horse embryos.

Development of horse embryos up to twenty two days after ovulation: observations on fresh specimens.

Van Niekerk & Gerneke (1966) first showed that in mares several unfertilized ova can be found in the oviduct at any one time. This has been confirmed by Webel, Ellicott & Dziuk (1970), Thompson (cited by Allen, 1970), Betteridge & Mitchell (1972) and Steffenhagen, Pineda & Ginther (1972) and has also been shown to occur in donkeys (Bielanski & Zapletal, 1968). It seems highly probable that these ova originate from previous normal cyclic ovulations and persist in the oviduct for several months, but the evidence for this has so far been circumstantial in that persistence of individually marked ova has not been followed. During studies on the origin of accessory CL, we obtained direct evidence in two mares that individual ova were retained in the oviduct for periods of at least 24 days and 52 days. Three ova had been marked naturally by sperm penetration without fertilization at isolated matings and then, after these intervals, were recovered from the oviducts at slaughter. Mare No. 1 was an aged, parous, 200-kg pony which was subjected to laparotomy during anoestrus on 10th November, 1972. The oviducts were flushed (Betteridge & Mitchell, 1972) to remove old ova and 3200 i.u. PMSG (Gestyl, Organon) was injected into the ovarian stroma in an attempt to induce superovulation. No response was detected by rectal palpation during the next 14 days. Twice daily subcutaneous injections of 500 μg synthetic LH-releasing hormone (LH-RH, Ayerst) were given for 5 days beginning on 29th January 1973, together with daily intramuscular injections of 12,500 i.u. PMSG (Ayerst) on the last 2 days in a further attempt to induce superovulation. Again, no palpable response was detected. No critical examinations for oestrus were made thereafter until 5th April, when daily teasing with a stallion and thrice weekly inspection of the cervix per vaginam were commenced. Frequent rectal pal¬ pation of the ovaries was initiated each time signs of impending oestrus were seen and were continued until oestrus ended. Particular emphasis was placed on detection of ovulation by recording the consistency and dimensions of developing follicles. Five oestrous periods of 5, 3, 3, 6, and 4 days were recorded, beginning at intervals of 19, 19, 18 and 20 days, respectively. Only a single mating, on the 2nd day of the last oestrus, was permitted. At no time did the ovaries show any marked enlargement, maximum follicle size being estimated at 2 cm. Ovulations were detected only twice (second oestrus, right side ; third oestrus, left side). Following the fifth oestrus, the mare was consigned to a virus

/pdf/direct-evidence-of-retention-of-unfertilized-ova-in-the-38ze96hebr.pdf

D. Mitchell

Papers

Collection, description and transfer of embryos from cattle 10--16 days after oestrus.

Development of horse embryos up to twenty two days after ovulation: observations on fresh specimens.

Direct evidence of retention of unfertilized ova in the oviduct of the mare

Sexing two-week old bovine embryos by chromosomal analysis prior to surgical transfer: Preliminary methods and results

Maternal progesterone levels as evidence of luteotrophic or antiluteolytic effects of embryos transferred to heifers 12–17 days after estrus