Detection of histocompatibility-Y antigen: identification of sex of pre-implantation ovine embryos.

Indirect immunofluorescence was used to detect the presence of a male-specific protein (H-Y antigen) on pre-implantation ovine embryos. Eight-cell to blastocyst stage embryos were surgically collected from superovulated ewes, classified as fluorescent (H-Y positive) or non-fluorescent (H-Y negative) and either transferred to recipient ewes or karyotyped to confirm embryonic sex. H-Y antigen was detected on eight-cell through blastocyst stage embryos. Overall, 88% (50/57) of the embryos (eight-cell to early blastocyst stage) classified as H-Y positive or H-Y negative were male and female, respectively. Survival after transfer of embryos subjected to the H-Y antigen assay was high (63%), which supports the use of this procedure in conjunction with embryo transfer in sheep to produce pregnancies in which sex of the fetus is known.

Buffalo and cattle hybrid embryo development is decreased by caffeine treatment during in vitro fertilization.

Water buffalo are renowned for difficulties in the implementation of assisted reproductive technologies, with both males and females being problematic. In this study, we used cattle oocytes to assess the effect of treatments with heparin and caffeine on buffalo spermatozoa and subsequent fertilization and embryo development in vitro. There was no significant difference between buffalo and bovine spermatozoa in the events associated with fertilization. Fertilization of cattle oocytes with buffalo spermatozoa resulted in 7.8% of oocytes developing into hybrid embryos. A difference in the developmental capability of hybrid embryos compared with the cattle control was observed. This has not been previously reported. The subsequent transfer of a limited number of hybrid embryos did not produce a viable pregnancy. However, control treatments in this experiment also failed to achieve pregnancy, so objective data is not available to provide conclusions about the developmental competence of the buffalo and cattle hybrid embryos. Optimal spermatozoa capacitation treatments achieved 61% fertilization and 21% zygote cleavage into two cell embryos. There was no significant difference in fertilization or development due to heparin or spermatozoa concentrations. However, treatment of buffalo and cattle spermatozoa with caffeine significantly decreased embryo cleavage but also tended to decrease embryo development to the blastocyst stage. These studies suggest that problems with reproduction in buffalo may reside with biological mechanisms associated with the oocyte that are often complicated by poor male reproductive performance. Selection for bull fertility would prevent some of these complications.

Competition between split and nonmanipulated embryos in the production of identical piglets.

The effect of nonmanipulated embryos on in utero survival of split porcine embryos was examined in this study. Previously, only limited success in the production of identical twin piglets has been reported. Embryos were collected from slaughtered donors (4 to 7 d post estrus) and were either split with the aid of a micromanipulator or left as whole embryos. Monozygotic pairs of split embryos were then surgically transferred to recipients with a complement of either split or nonmanipulated embryos. A total of 217 split embryos and 60 nonmanipulated embryos were transferred to 19 recipients. Nine of these recipients farrowed. In the two recipients that received only split embryos and farrowed, 31% of the split embryos survived to term, including two sets of monozygotic twins. In the remaining seven recipients, only 10% of the split embryos that were transferred along with nonmanipulated embryos survived to farrowing. This difference in split embryo survival (31 vs 10%) was significantly different (P<0.005). Sixty-nine percent of the nonmanipulated embryos survived to term in recipients that maintained pregnancy. Data presented in this study suggest that competition occurs between split and nonmanipulated embryos transferred to the same uterine environment.

Low doses of oxytocin can induce foaling at term.

Levels of the major circulating metabolite of prostaglandin F-2 alpha, 13, 14 dihydro-15-oxo-prostaglandin F-2 alpha (PGFM) were measured during the induction of foaling using small (2.5-10 iu) intravenous doses of oxytocin. PGFM levels rose rapidly in all animals within 15 min of injection and were associated with typical signs of second stage labour. Because these small doses of oxytocin are effective in successfully triggering parturition it is suggested that higher doses (40-120 iu) used to induce birth in other studies are unnecessary and could be potentially dangerous to the foetal foal.

Production of ovine chimeras by inner cell mass transplantation.

Ovine chimeras were produced by micro-injection of isolated inner cell masses (ICM) into recipient blastocysts. Inner cell masses were isolated by immunosurgery. A total of 57 chimeric embryos was produced, 52 of which were transferred to recipient ewes. Thirty-seven live lambs were born, of which 15 were determined to be chimeric on the basis of blood type analysis. One lamb, although not a blood chimera, exhibited overt signs of chimerism. An additional six lambs were determined to have developed solely from the injected ICM. The rate of chimerism in live lambs was 43% (16/37) while the survival rate of injected ICM was 59% (22/37). The method presented allows the production of relatively large proportion of viable, chimeric embryos without the use of an intermediate recipient.

Production of sheep-goat chimeras by inner cell mass transplantation.

Embryos were surgically flushed from goats and sheep on d 6 and 7, respectively, following the first day of estrus (d 0). After enzymatic removal of the zonae pellucidae, inner cell masses were isolated from caprine blastocysts by immunosurgery. The intact inner cell masses were injected into ovine blastocysts with the aid of a micromanipulator. Twenty-two manipulated blastocysts were surgically transferred into 12 ovine recipients. Nine ewes gave birth to a total of 13 young (59% embryo survival). Ten were classified by serum electrophoretic assays or karyotypes as lambs, one as a kid, and two as interspecific chimeras.

Biotechnology. The key to improved animal production?

Biotechnology will influence the reproductive performance of cattle in a number of different ways. In animals breeding, embryo transfer has already had a significant impact on the way in which genetic selection of animals is performed. If other technologies such as embryo splitting, in vitro fertilization, cloning, and sex selection are superimposed on conventional embryo transfer, genetic gains will be achieved at an extremely rapid rate. The net result be a rapidly changing animal population in which genetic gains are maximized. Transgenic animals with altered genetic make-up will also have a dramatic effect on animal production. The general introduction of such animals will be slow and the cost to produce them will be enormous, as conventional selection and progeny testing will be needed to ensure that the "new" introduced genes are functional and do not have detrimental side effects. Simple, sensitive assays of hormones and proteins developed using biotechnology will aid in the monitoring of reproductive performance of animals.

Preliminary observations on reproduction in a female sheep-goat chimaera.

Four oestrous cycles of a female sheep-goat chimaera were monitored by using a vasectomised ram. The mean (+/- se) length of the cycle was 18.5 +/- 0.64 days with a range from 17 to 20 days. The chimaera was superovulated twice, bred to fertile rams, and the embryos recovered by laparotomy 13 or five days after oestrus, so that karyotype analysis could reveal the genotype of the oocyte. After the first superovulation one ovine day-13 embryo was collected; two fragments of another embryo (or embryos) were also collected, but readable chromosome spreads were not obtained from these embryos or from the two four-cell embryos that were collected five days after the second superovulation. Two surgical embryo transfers to the chimaera resulted in pregnancies. The first transfer involved an eight-cell ovine embryo and two caprine morulae and ended in the abortion of an ovine fetus between days 110 and 130. The second pregnancy occurred after the transfer of two ovine and two caprine morulae. A healthy lamb was born on day 147 of pregnancy. Both placentae had small numbers of cotyledons. A histological evaluation of the cotyledons revealed an abnormal placentome structure in the first pregnancy but not in the second.

Production of monozygotic (identical) horse twins by embryo micromanipulation.

The blastomeres of 192- to 8-cell embryos recovered surgically 1-3 days after ovulation from 23 Pony mares were mechanically separated and inserted, in various combinations, into evacuated pig zonae pellucidae to make 27 'half' and 17 'quarter' micromanipulated embryos. These were embedded in agar and cultured in vivo in the ligated oviducts of ewes for 3.5-5 days to allow development to the late morula/early blastocyst stage. Subsequent surgical or non-surgical transfer of 13 'half' and 17 'quarter' embryos to mares resulted in 10 established pregnancies, including 2 monozygotic pairs. Surgical transfer to mares that had not been recently used as donors of embryos was more successful (10/20) than surgical or non-surgical transfer to recently operated mares (0/10).

The role of the fetal gonads and placenta in steroid production, maintenance of pregnancy and parturition in the mare.

The effects of fetal gonadectomy on steroid production and the maintenance of pregnancy in the mare were studied. Removal of the fetal gonads resulted in an immediate fall in maternal plasma concentrations of conjugated and unconjugated oestrogens whereas progestagen levels remained unchanged. Hormone profiles in mares carrying sham-operated fetuses remained similar to those in unoperated control mares. Plasma levels of 13,14-dihydro-15-oxo-PGF-2 alpha (PGFM) were much lower, and uterine contractions weaker, during labour in mares carrying gonadectomized foals than in control mares. Pregnancy was maintained until parturition at term in the mares carrying gonadectomized fetuses. However, 3 of the 4 gonadectomized foals were dysmature and died during or soon after birth. The biosynthetic pathways involved in the production of oestrogens by the feto-placental unit and the possible role of oestrogens in fetal development in the pregnant mare are discussed.

The use of synthetic prostaglandin analogue (fluprostenol) to induce foaling.

The synthetic prostaglandin (PG) analogue fluprostenol was used to induce parturition in mares and its mode of action was investigated by measuring endocrine changes before and during the induction period. Progestagens and unconjugated oestrogens showed little change during the induction period, but two different patterns in plasma PGFM levels were observed. The first was seen when foaling occurred within 90 min of injection; PGFM levels rose soon after injection and peaked during the maximum expulsive stage of labour, thus resembling events during natural foaling. The second occurred when foaling took longer than 90 min, and in these mares PGFM levels rose at various times after injection and peaked well before the onset of the expulsive stage of labour. It is suggested that these differences reflect the hormonal readiness of the mares to foal. Other procedures, such as rupture of the allantochorion, and dilatation of the cervix and injection of fluprostenol into the allantois, produced no uterine activity and did not stimulate labour or PGFM release.

Survival of sheep x goat hybrid inner cell masses after injection into ovine embryos.

Modified blastocyst injection techniques were used to inject immunosurgically isolated sheep x goat hybrid inner cell masses (ICM) into ovine blastocysts, with subsequent transfer of composite embryos to ovine recipients. Hybrid embryos were collected from does artificially inseminated with Barbados ram semen. A total of 13 live and 2 aborted offspring resulted from the 34 composite embryos transferred to recipient ewes (38% embryo survival). Of the 15 offspring, 4 exhibited phenotypic hybridism and 2 (13%) of these were determined to be hybrid mean value of -sheep chimeras by karyotype, serum protein and isoenzyme analyses, and fiber identification. Each of the 4 was produced by an injection procedure that involved damage of the ovine host ICM. One additional offspring was unusual in appearance, but the presence of hybrid cells was not proven. Similarly, caprine ICM were immunosurgically isolated and injected into ovine blastocysts that were then transferred to ovine recipients. Of the 13 composite embryos transferred, 12 offspring were produced (92% embryo survival). Eleven were overt goat mean value of -sheep chimeras and, of these, 7 were also blood chimeras. The hybrid ICM was shown to be capable of contributing to normal embryonic and fetal development after injection into an ovine blastocyst but may be less likely to be incorporated with the ovine host ICM than is the caprine ICM.

Breed differences in circulating equine relaxin.

Equine relaxin has been previously determined in a small number of pregnant Thoroughbred mares. To better define the normal pregnancy pattern of relaxin, the current study reports on a much larger number of mares. It also was designed to determine if all equids have the same gestational pattern of relaxin secretion. Plasma samples were collected weekly in 24 Standardbred mares, every 7-10 days in 10 pony mares, and daily in late pregnancy from 16 burros. Standardbreds had higher concentrations of relaxin than that reported for Thoroughbreds during most of gestation and did not exhibit the midpregnancy nadir in relaxin concentrations observed in Thoroughbreds. Relaxin concentrations in Standardbreds showed a small but steady decline from Day 150 until delivery. Pony mares had lower relaxin concentrations throughout pregnancy than other mares and had continuously increasing concentrations during gestation. Burros had relaxin concentrations intermediate to ponies and other mares in late gestation. Burros induced to foal with oxytocin showed a sharp increase in relaxin concentrations. No effect of the sex of the offspring was observed in relaxin profiles in Standardbred mares. Each of three Standardbreds with abnormal termination of pregnancy exhibited abnormally low relaxin concentrations at some point in the gestation prior to termination of the pregnancy. Thus, relaxin may be an indicator of placental functioning and used to assess at-risk pregnancies in mares.

Dehydroepiandrosterone synthesis by the fetal foal and its importance as an oestrogen precursor.

The gonads of the fetal horse were found to be relatively devoid of 3 beta-hydroxysteroid dehydrogenase and other enzymes which metabolize dehydroepiandrosterone (DHA). In short-term in-vitro incubation experiments fetal liver converted DHA to the potential equilin precursor, 7 alpha-hydroxy DHA. DHA was converted to oestrone when incubated with extracts of horse placenta but 7 alpha-hydroxy DHA was not converted to equilin. Levels of DHA measured in peripheral blood of mares throughout pregnancy paralleled those of equilin and oestrone, and DHA concentrations fell rapidly after fetal gonadectomy, as did those of equilin and oestrone. Administration of [4-(14)C]-DHA and [7-3H]DHA to the fetus resulted in the incorporation of both 14C and 3H into maternal urinary oestrone but neither isotope was present in urinary equilin. These findings confirm the role of fetal DHA as a precursor of oestrone, but do not support the previously suggested role of the fetal liver in the synthesis of equilin. They are, however, compatible with the hypothesis that equilin is formed via a pathway which diverges from the terpenoid steroid synthetic route before DHA.

Antibody response of ewes and does to chimeric sheep-goat pregnancy.

Antibody production was evaluated in 62 recipients of blastomere-aggregation sheep-goat embryos, including 23 multiparous ewes, 21 multiparous does, 16 primiparous does, and 2 virgin does. The reactivity of sera collected weekly after the embryo transfer surgery was compared to that of sera collected prior to the embryo transfer by means of 1) complement-dependent cytotoxicity tests against peripheral blood lymphocytes (PBLs) from the parents of the embryo(s) and from random-bred sheep and goats, 2) hemagglutination and hemolytic assays with red blood cells (RBCs) from the two sires of the embryo(s), and 3) assays with PBLs and RBCs following absorptions with RBCs and PBLs from the parents and offspring. Although cross-reactivity to ovine and caprine PBL antigens was present in the control sera of some recipients, xenogeneic immunization during pregnancy was detected in 20 of 30 recipients that experienced term pregnancy. The xenogeneic response involved the production of antibody that reacted with both PBLs and RBCs. Allogeneic responses to RBCs were not observed, but allogeneic responses to PBLs occurred frequently, beginning after the onset of the xenogeneic response in most recipients (98 +/- 28 vs. 57 +/- 15 days in ewes; 93 +/- 23 vs. 46 +/- 7 days in does; mean day of onset +/- SD). The onsets of the responses were examined in conjunction with data collected on fetal and placental chimerism to evaluate possible routes of immunization. The onsets of the allogeneic responses and the limited serum reactivity to third-party PBLs suggested that fetal lymphocytes leaking across the placenta immunized the recipients to parentally inherited polymorphic antigens. The xenogeneic responses were associated with placental chimerism and appeared to involve the recognition of a species-specific monomorphic antigen shared by PBLs and RBCs. Neither of the responses appeared to affect continuation of pregnancy to term.

Effects of chimerism in sheep-goat concepti that developed from blastomere-aggregation embryos.

Chimeric sheep-goat pregnancies were established in 24 ewes and 29 does by transferring 251 embryos, prepared by the blastomere-aggregation technique, to 52 ewes and 61 does. Fifteen does experienced early pregnancy failure; however, term offspring were delivered by 24 ewes (17 lambs, 3 kids, 6 chimeras) and 14 does (6 lambs, 9 kids, 6 chimeras). (Fetal classifications were based on phenotype, red blood cell isozymes, and lymphocyte antigen expression). RIAs for ovine and caprine placental lactogen detected chimerism in the binucleate cell population of the trophoblast throughout the pregnancies of 2 ewes and 7 does; these pregnancies resulted in the birth of 12 healthy offspring. Histological examinations of intact placentomes from 2 of these recipients revealed a continuous cellular trophoblast apposed to a syncytium as in normal placentas. Chimerism was detected electrophoretically in the membranes of the placentas with binucleate cell chimerism and in 17/28 of the other placentas. Data collected on placental lactogen production, chimerism in the conceptus, and placental morphometry were examined with respect to the stages of the blastomeres aggregated to form the chimeric embryo and with respect to fetal status at delivery. For comparison, analogous data were collected on sheep-goat concepti that developed from embryos prepared by inner cell mass transplantation.

Identification of a male-specific histocompatibility protein on preimplantation porcine embryos.

An indirect immunofluorescence assay was used to detect the presence of male-specific protein(s) on various stages of preimplantation porcine embryos. Embryos were collected at slaughter from the reproductive tracts of day-2.5, -4, -5, -6, and -8 (day 0 = first day of estrus) sows and gilts. Embryos were placed in medium containing an anti-male primary antibody, washed, and transferred to culture drops containing a fluorescein isothiocyanate (FITC)-labeled secondary antibody. Embryos were classified as either fluorescent (H-Y positive) or nonfluorescent (H-Y negative), transferred to coded drops, and karyotyped to examine sex chromosomes. A total of 91 eight-cell to blastocyst stage embryos were evaluated; of these, 46% were classified as fluorescent and 54% as nonfluorescent. Of readable metaphase spreads (65%) from these embryos, 81% (48 of 59, P less than 0.005) were correctly sexed by immunological detection of the male-specific antigen. Although 13% (2/15) of four-cell embryos evaluated were classified as fluorescent, the accuracy with which embryos at this stage were sexed by detection of H-Y antigen was not different from 50%. Fifty percent of eight-cell embryos were classified as H-Y positive with 78% of embryos correctly sexed. It was concluded that the eight-cell embryo is the earliest stage of development for which there is evidence for expression of H-Y antigen. Detection of the male-specific protein was difficult at the expanded blastocyst stage.