Cleavage, development and competence of sheep embryos fertilized by intracytoplasmic sperm injection and in vitro fertilization

More abnormal fertilization has been found in sheep oocytes after intracytoplasmic sperm injection (ICSI) than after in vitro fertilization (IVF). Although the birth of a normal lamb has been reported, the efficiency of blastocyst production is low. We therefore evaluated the cleavage, development and viability of sheep embryos obtained from ICSI, IVF and sham injection. In vitro matured oocytes either injected or inseminated with spermatozoa were assessed for cleavage 1 and 4 d after injection or insemination, and for development to blastocyst after 7 d of culture. A total of 699 oocytes was injected (ICSI); 198 (30.6%) were activated and 55 (8.5%) developed to the blastocyst stage. Of the 17 recipient ewes with 1, 2, 3 or 4 embryos, 15 (88.2%) were pregnant on Day 18; of these 17 recipients, 7 (41.1%) and 6 (35.2%) ewes remained pregnant on Days 45 and 110, respectively. Two normal lambs were born, one ewe died on Day 110 with 2 normal male fetuses, another ewe aborted on Day 90 and 4 pregnancies were maintained. A total of 517 oocytes was inseminated (IVF); 296 (62%) were activated and 90 (18.8%) reached the blastocyst stage. A total of 19 ewes received 1, 2, 3 or 4 embryos; of these, 13 (68.4%) were pregnant on Day 18, 8 (42.1%) ewes remained pregnant on each of Days 45 and 110. Three ewes delivered 5 lambs. Five pregnancies were maintained. A total of 156 oocytes was sham injected, 38 (24.3%) were activated and no blatocysts were obtained after culture. The results of this study showed that blastocysts obtained after ICSI are potentially viable and are not a result of parthenogenesis.     

Screening of some variables influencing the results of embryo transfer in the ewe. I. Five-day-old embryos

Rasa Aragonesa ewes (n = 89) received 2 embryos on Day 6 of the estrous cycle (Day 0 = estrus) from 46 donors of the same breed that had been superovulated with FSH-p. The influence of several variables on fertility and prolificacy after transfer was studied by discriminant analysis. Our results showed that the main variables that contributed to a high fertility rate were the degree of synchrony (better outcome if donors come into estrus later than the recipients); Fluorogestone acetate (FGA) to estrus interval and interval from previous lambing in the recipients, ovulation rate of the donors and recipients (better if superior to the mean); prolificacy of recipients in the previous lambing; and difference in developmental stage of the pair of transferred embryos (better if inferior to the mean). The main variables affecting prolificacy were the ovulation rate and weight of the donors and progesterone concentrations of the recipients (better if lower than the mean); age of the donors and difference in progesterone concentrations between donors minus those of the recipients (better if higher than the mean). The percentage of ewes correctly classified into lambing or not lambing status was 73% (P < 0.001) and that of the ewes correctly classified as lambing 1 or 2 lambs was 81.8% (P < 0.001). Whether or not the criteria we have established for optimum transfer results are applicable to conditions other than our own still needs to be confirmed.     

Embryonic survival in the uterus of ewes inseminated at the uterotubal junction on Day 32 post partum

Experiments were conducted to determine 1) if pregnancy initiated on Day 32 post partum would be maintained until lambing, 2) if there is a difference in the ability of the previously gravid or nongravid uterine horn to maintain pregnancy, and 3) if season has an effect on embryo loss. Estrus was induced in ewes on Day 32 post partum. At estrus, ewes were inseminated surgically at the uterotubal junction and assigned to the following groups: 1) inseminated at estrus and laparotomized on Day 3 to collect embryos for determination of fertilization rate (C), 2) inseminated in the previously gravid uterine horn (PG), 3) inseminated in the previously nongravid uterine horn (NG), and 4) inseminated when both horns were previously gravid (BG). Ewes pregnant in the PG, NG and BG groups were allowed to lamb. Conception rate in Group C at embryo collection was 70%. Embryo loss, based on concentrations of progesterone at Day 18 post insemination, was 43, 19 and 18% in the BG, NG and PG group, respectively. The high embryo loss in Group BG occurred only during the breeding season. Only 24% of the ewes that had been inseminated lambed. This was due to the prepartum loss of embryos and fetuses (47, 48 and 33% in Group BG, NG and PG, respectively. In conclusion, the detrimental effects of the uterus on embryo survival was evident within 18 d post insemination in Group BG (breeding season), and embryo loss prior to lambing was high in all the treatment groups (both seasons).     

Screening of some variables influencing the results of embryo transfer in the ewe. Part II: two-day-old embryos

Each of sixty Rasa aragonesa ewes received two embryos on Days 2-3 of the estrous cycle (Day 0=estrus) from 27 donors of the same breed that were superovulated with pFSH. The influence of several variables on fertility and prolificacy after transfer was studied by discriminant analysis. Our results showed that the main variables contributing to higher fertility were: in the donor-recipient couple, degree of estrus synchrony between them (better if donors were in estrus before recipients); in recipients, interval from FGA withdrawal to estrus onset, prolificacy in the previous lambing, age (all, better if inferior to the mean) and interval from the previous lambing (better if superior to the mean); in donors, ovulation rate (better if lower than the mean); and in embryos, developmental stage (better if superior to the mean). Likewise, the main variables contributing to higher prolificacy were: in donors, body condition score (better if higher than the mean) and weight (better if inferior to the mean); and in recipients, plasma progesterone concentration at transfer (better if inferior to the mean). The percentage of ewes correctly classified as lambing or not was 71.7% (P<0.01), and 72.5% of the ewes were correctly classified as having one or two lambs (P<0.05). Whether the criteria we have found for optimum results after transfer are applicable or not to conditions other than ours will need to be confirmed.     

Maintenance of the corpus luteum after uterine transfer of trophoblastic vesicles to cyclic cows and ewes

One or two trophoblastic vesicles (0.4-2 mm diam.) from cow (Day 14) or ewe (Day 11-13) embryos without their disc were transferred, after culture for 24 h, into recipients. Each vesicle was transferred into the uterine horn ipsilateral to the CL by the cervical route in heifers and surgically in ewes on Day 12 of the oestrous cycle. In cows, daily measurements of plasma progesterone concentrations and checks for return to oestrus showed that the CL was maintained in 8 out of 12 recipients. These 8 cows had 25- to 37-day cycles while 4 recipient heifers returned to oestrus normally. Three recipients with an extended cycle were slaughtered. The dissected uterus showed that trophoblastic vesicles had developed in the uterine horns. In ewes, the serum progesterone curve, determined in each recipient, showed that the CL was maintained in 7 out of 12 recipients. These 7 ewes had 20- to 54-day cycles and the other 5 ewes had a normal cycle of 15-19 days comparable to that of 17.0 +/- 0.5 days for the 6 control ewes. Whenever the CL was maintained, high blood progesterone levels were followed by rapid luteolysis. In cattle and sheep, therefore, a trophoblastic vesicle transferred into the uterus can develop in vivo, secreting the embryonic signals when there is no embryonic disc control and transforming the cyclic CL into a CL of pregnancy in about 60% of the cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Progestin implants can rescue demi-embryo pregnancies in goats: a case study

Survival after transfer of demi-embryos (i.e., half-embryos produced by embryo splitting) to recipients usually is lower than survival after transfer of intact embryos. Reduced survival after demi-embryo transfer could be due to loss of viability after splitting, failure of a viable demi-embryo to prevent corpus luteum (CL) regression in the recipient female, or a combination of factors. From a retrospective analysis of pregnancy and embryo survival rates after demi-embryo transfer in sheep and goats, we report the rescue of caprine demi-embryo pregnancies in which CL regression occurred at the end of diestrus despite the presence of a viable conceptus in the uterus with progestin implants. Day 5 or 6 morulae and blastocysts were flushed from superovulated ewes and does and split into demi-embryos of approximately equal halves. Demi-embryos were either transferred fresh to synchronized recipients of the homologous species or frozen in liquid nitrogen. Approximately half of the recipient does and ewes were treated with norgestomet implants on Day 10 of the embryo transfer cycle and again 2 wk later. Serum collected on Day 25 from recipients with implants was assayed for progesterone to determine if a CL of pregnancy had been maintained. Pregnancy was diagnosed by ultrasonography on Day 35 of gestation. Corpus luteum regression occurred despite the presence of a viable conceptus in the uterus in 6 of 55 progestin-treated caprine demi-embryo recipients and in 0 of 66 ovine demi-embryo recipients. Five of the caprine pregnancies were maintained to term with norgestomet implants and produced 5 live kids. The sixth fetus, which was carried by a progestin implant-treated 8-mo-old doeling, died at approximately 50 d of gestation. These results suggest that, at least in goats, some demi-embryos may provide inadequate signaling for maternal recognition of pregnancy, and such pregnancies can be rescued with progestin treatment to the doe.     

Seroconversion of recipient ewes after transfer of ova exposed to Brucella ovis in vitro

Zona pellucida-intact ova collected from ewes seronegative to Brucella ovis were exposed in vitro to B ovis and washed 10 times in medium that contained no antibiotics. After exposure and washing, nontransferable ova were cultured for isolation of Brucella , and the viable ova were transferred into seven B ovis seronegative ewes. No pregnancies resulted, thus recipient ewes were bred during the next breeding season, and blood samples were collected for bacteriological and serological examination until one month after lambing. Brucella ovis was isolated from all of the nontransferable ova, indicating that the transferred ova had viable organisms adhered to them. Although no recipient was found to be pregnant at Day 45, all seven ewes responded to the transferred ova by producing anti-Brucella antibodies. With the exception of a ewe that was euthanized early in the project due to a traumatic injury, all recipients lambed normally during the following breeding season. Brucella was not found in any sample collected from ewes or lambs. However, ELISA titers for B ovis remained in the suspicious range and a ewe was positive on the CF test within 2 wk of lambing.     

Interspecific transfer of IVM IVF-derived red sheep (Ovis orientalis gmelini ) embryos to domestic sheep (Ovis aries )

Two embryo production methodologies were investigated to generate Red sheep embryos for use in an interspecific embryo transfer program. In Experiment 1, 4 multiparous female Red sheep (Ovis orientalis gmelini ) were implanted with CIDR type G devices for 11 d. Forty-eight hours prior to CIDR removal, a total of 22.5 mg bid of FSH-P was administered over a 3-d period. Laparoscopic embryo collection was performed 5 d post breeding, and embryos were transferred to domestic recipient ewes (Ovis aries and Ovis orientalis musimon ). In Experiment 2, 7 nulliparous female Red sheep were implanted with CIDR devices and injected with 200 IU of PMSG and 25 mg of FSH-P on the 8th day of implant insertion. At 60 to 70 h post PMSG/FSH-P treatment, follicular oocytes were aspirated laparoscopically. The recovered oocytes were matured in M199 (with fetal calf serum, FSH, LH, penicillin and streptomycin) at 39 degrees C in a humidified atmosphere containing 5% CO(2). At 24 h oocytes were fertilized with frozen-thawed semen at a concentration of 1.6 x 10(6) sperm/ml. The ova/embryos were placed in CR2 or BOEC culture medium at 20-22 h post IVF. Following 3 to 4 d in culture, embryos were transferred laparoscopically to the uterine horn of synchronized recipients. In Experiment 1, 4 embryos and 6 UFO were collected from 2 embryo donors, respectively. Two embryos were transferred with the aid of a laparoscope to each of 2 Rambouillet recipients, one of which gave birth to a healthy Red sheep lamb at 158 d of gestation. In Experiment2, a total of 62 oocytes was collected from 7 oocyte donors; 16 developed to the 16- to 32-cell stage and were transferred to 8 recipients. Three of these IVM-IVF embryos were transferred laparoscopically to 2 Mouflon recipients, resulting in no pregnancies. Thirteen IVM-IVF embryos were transferred to 6 Rambouillet recipients. Each of these gave birth to a single healthy Red sheep lamb. Gestation lengths of the 3 IVM-IVF lambs ranged from 152 to 162 d. This research demonstrates that when using compatible species IVM-IVF technology in conjunction with interspecific ET can lead to the production of live offspring and can be used to propagate exotic ovine species.     

Validation of a new pregnancy-associated glycoprotein radioimmunoassay method for the detection of early pregnancy in ewes

The aim of the current study was to describe the use of a pool of different antisera raised against pregnancy-associated glycoproteins (PAGs; purified from both ovine and caprine placentas) for early pregnancy diagnosis in ovine species. Sixty-three pluriparous Sarda ewes (Ovis aries) were synchronized. Blood samples were withdrawn on Days 18, 24, 26, 28, 30, and 50 after mating. These samples were assayed for progesterone (radioimmunoassay [RIA] including an extraction step) and for pregnancy-associated glycoproteins (RIA-706 and RIA-srPool). Progesterone concentrations were under 1.0 ng/mL in all nonpregnant Sarda ewes. In pregnant ewes, mean progesterone concentrations ranged from 2.4 ng/mL (Day 24, single pregnancies) to 4.4 ng/mL (Day 28, multiple pregnancies). During all periods of examination, PAGs remained lower than 0.8 ng/mL in nonpregnant ewes. On Day 18 of pregnancy, PAG concentrations could be detected in 26 of 43 (60.5%) and in 41 of 43 (95.3%) pregnant ewes using the RIA-706 and RIA-srPool methods, respectively. From Day 24 to Day 50, using both RIA methods, PAGs could be detected in all pregnant ewes. On Day 24, the best threshold for pregnancy diagnosis was obtained by use of RIA-srPool, maximal concentration in nonpregnant ewes being 0.3 ng/mL and minimal concentration in pregnant ewes being 4.8 ng/mL. In general, progesterone and PAG concentrations were higher in multiple pregnancies than in single pregnancies. However, because of large individual variations, single pregnancies could not be differentiated from multiple pregnancies.     

Successful pregnancies after transfer of embryos recovered from ewes induced to ovulate 24-29 days post partum

After lambing in late November, oestrus and ovulation were induced by using a CIDR device and PMSG in early weaned (N = 13) or lactating (N = 14) Border Leicester x Scottish Blackface ewes between 23 and 29 days after parturition. Ewes were intrauterine inseminated under laparoscopic visualization 54-55 h after CIDR-device withdrawal and eggs recovered on Day 3 of the cycle. Ovum recovery and fertilization rates were higher in lactating than in early weaned ewes, with fertilization being achieved as early as 24 days post partum in both groups. Of the 7 early weaned and 11 lactating ewes yielding eggs, fertilization occurred in 4 and 7 ewes respectively. A total of 20 embryos were transferred to the normal uterine environment of 15 recipient ewes in which the interval from parturition was greater than 150 days. Pregnancies were successfully established in 9 recipient ewes, resulting in the birth of 10 viable lambs. Prolactin concentrations were significantly higher (P less than 0.001) in lactating than in early weaned ewes throughout the study. Nevertheless, normal luteal function (as assessed by daily progesterone concentrations) was exhibited by 12 of 14 lactating and 8 of 13 early weaned ewes. Two post-partum donors in which the corpora lutea completely failed to secrete progesterone yielded fertilized eggs which developed to term when transferred to a normal uterine environment. The results show that sheep oocytes can be fertilized using laparoscopic intrauterine insemination as early as 24 days after parturition and that the resulting embryos are viable when recovered on Day 3 after oestrus and transferred to a normal uterine environment.     

Increasing reproductive rates in tropical sheep by means of embryo transfer

One to three embryos were transferred to three groups each of 12 Kenya Merino ewes to establish if uterine capacity is a limiting factor to reproductive performance in this breed of sheep, in a tropical environment. A fourth group of 12 ewes received three embryos following superovulation. Multiple transfers increased the number of lambs born per pregnant ewe. However, although superovulation significantly (P<0.01) increased endogenous progesterone levels in Group 4 recipient ewes, it did not improve either their conception or lambing rates. Peri- and post-natal losses increased with the number of embryos trnasferred and with the litter size. Consequently, the same number of lambs were weaned per recipient ewe in all four groups. It is concluded that although the uterine capacity of the Kenyan Merino ewes is higher than their natural ovulation rates require, increasing the litter size will not necessarily increase the number of lambs weaned.     

Donor and recipient ewe factors affecting in vitro development and post-transfer survival of cultured sheep embryos

Donor and recipient factors were assessed during development of embryos following superovulation, collection at the pronuclear and two-cell stage, culture in Synthetic Oviduct Fluid medium for 5 days and twin transfer into synchronised recipients to elucidate what factors affect embryo development and post-transfer survival. In particular, the administration of exogenous progesterone to recipients using an intravaginal CIDR^TM device immediately following embryo transfer was investigated.

From 138 embryos collected from 30 donor ewes, 75% (103) were of transferable quality following culture, of which 100 were transferred to 50 recipients. There was significant variation (P < 0.001) in embryo development to the blastocyst stage between different donor ewes, but this was not related to the donor ovulation rate. At ultrasound sonography (approximately Day 60 of pregnancy), 58% of recipients were pregnant and 42% embryos had survived. Donor ovulation rate was related to embryo survival (P < 0.05) after transfer; the survival rate of embryos from ewes with high ovulation rates was lower than that of embryos from ewes with low ovulation rates. Exogenous progesterone supplementation following transfer did not affect embryo survival, rate of embryo development or plasma progesterone levels. In general, the results from this study suggest that factors other than efficacy of embryo culture can affect the outcome of embryo survival following transfer and that, where possible, these factors should be considered and balanced in experimental designs.     

Protostrongylid parasites and pneumonia in captive and wild thinhorn sheep (Ovis dalli)

We describe health significance of protostrongylid parasites (Parelaphostrongylus odocoilei and Protostrongylus stilesi) and other respiratory pathogens in more than 50 naturally infected Dall's sheep (Ovis dalli dalli) from the Mackenzie Mountains, Northwest Territories (1998-2002) as well as in three Stone's sheep (O. d. stonei) experimentally infected with P. odocoilei (2000-2002). Histological lesions in the brain and distribution of P. odocoilei in the muscles of experimentally and naturally infected sheep were consistent with a previously hypothesized "central nervous system to muscle" pattern of migration for P. odocoilei. Dimensions of granulomas associated with eggs of P. odocoilei and density of protostrongylid eggs and larvae in the cranial lung correlated with intensity of larvae in feces, and all varied with season of collection. Prevalence of P. stilesi based on the presence of larvae in feces underestimated true prevalence (based on examination of lungs) in wild Dall's sheep collected in summer and fall. Similarly, counts of both types of protostrongylid larvae in feces were unreliable indicators of parasitic infection in wild Dall's sheep with concomitant bacterial pneumonia associated with Arcanobacterium pyogenes, Pasteurella sp., and Mannheimia sp. Diffuse, interstitial pneumonia due to P. odocoilei led to fatal pulmonary hemorrhage and edema after exertion in one experimentally infected Stone's sheep and one naturally infected Dall's sheep. Bacterial and verminous pneumonia associated with pathogens endemic in wild Dall's sheep in the Mackenzie Mountains caused sporadic mortalities. There was no evidence of respiratory viruses or bacterial strains associated with domestic ruminants, from which this population of wild sheep has been historically isolated.     

Pregnancy diagnosis and assessment of fetal numbers in the ewe in a commercial setting

Jugular plasma progesterone concentrations were used to accurately predict open ewes (96 +/- 3%) in early pregnancy, but they less accurately predicted subsequent lambing especially during the late breeding season and most of the seasonal anestrus. Progesterone values clearly indicated that 500 I.U. of P.M.S.G. elevated ovulation rate in synchronized ewes, but did not clearly indicate fetal numbers. During late pregnancy (88-108 days), abdominal palpation, doplar ultrasound and serum progesterone analysis were equally efficacious in predicting lambing (86 +/- 9.8%, 90 +/- 9.0% and 87 +/- 4.1%, respectively), but a high percentage of ewes lambed that were diagnosed as nonpregnant (30 +/- 15.0%, 48 +/- 17.3% and 25 +/- 8.4%, respectively). Accuracy of the serum progesterone test improved the later the test was performed, although considerable individual overlap existed. Progesterone values for ewes bearing 1, 2, or greater than 2 fetuses at 94 to 95 days of gestation differed (5.5 +/- 0.3, 8.0 +/- 0.4 and 12.4 +/- 2.1, respectively (P < 0.05), whereas at 103 to 108 days values for ewes carrying two or more fetuses did not differ.     

Interspecies pregnancy of Spanish ibex (Capra pyrenaica) fetus in domestic goat (Capra hircus) recipients induces abnormally high plasmatic levels of pregnancy-associated glycoprotein

Interspecies embryo transfer could be a valuable tool in preservation programs of endangered species. In this work the results of both interspecific-monospecific (ibex-in-goat) and interspecific-bispecific (mixed-species; ibex+goat-in-goat) embryo transfers in the capra genus are reported. The aim of this work was to compare the PAG plasmatic profiles occurring in these interspecific gestations to those encountered in normal (i.e. intraspecies) pregnancies of Spanish ibex and domestic goat. Spanish Ibex females were superovulated with 9 mg NIADDK-oFSH-17 and embryos were surgically collected 5.5 d after estrus. Two embryos were transferred per recipient. Domestic goat recipients were previously mated either to vasectomized domestic bucks (n=17 females; interspecific-monospecific gestations) or to fertile ones (n=9 females; interspecific-bispecific gestations). Intraspecific pregnancies were obtained by natural mating between males and females of the same species (Spanish ibex: n=6; domestic goat: n=1). Pregnancy rate diagnosed by progesterone was low in both interspecific-monospecific (7/17) and interspecific-bispecific (3/9) transfers. None of the monospecific (0/7) and 2 (2/3) of the bispecific established pregnancies developed to term. Ibex-in-ibex PAG profile showed 2 similar peaks of 60 to 70 ng/mL on Days 34 and 153 of pregnancy, while goat-in-goat had the maximum value (60 to 70 ng/mL) at Day 50, decreasing slightly afterwards until parturition. Mixed-species gestations (ibex+goat in goat) showed a first peak of 500 to 1000 ng/mL on Day 70 and a second one (200 to 500 ng/mL) on Day 140 of pregnancy. Four ibex-in-goat gestations that terminated with the expulsion of dead fetuses at Days 110 to 170 had their maximum PAG values (100 to 700 ng/mL) on Days 60 to 90. We conclude that it is possible to achieve pregnancies after transfer of ibex embryos into domestic goats, but this requires a great change of the PAG profiles, which increase significantly. Live ibex kids can be produced when embryos from both species share the uterus. This is the first report of successful interspecific pregnancies in the capra genus.     

An epizootic of Mycoplasma ovipneumoniae infection in captive Dall's sheep (Ovis dalli dalli)

In late spring of 1986, 10 of 23 Dall's sheep (Ovis dalli dalli) at the Metropolitan Toronto Zoo were moved to a new exhibit, where all developed severe respiratory signs refractory to anthelmintic and antibiotic therapy. In July, two animals died with chronic active bronch-pneumonia, and a third was euthanized because of pneumonia several months later. Bacteria were not isolated from the lungs of the first, steptococci and Pasteurella hemolytica were isolated from the other two, respectively; Mycoplasma ovipneumoniae was isolated from both. Pulmonary lesions in all three sheep were consistent with Mycoplasma sp. infection. Nasal swabs of the remaining animals yielded no consistent bacterial isolates; however, four of eight sheep were positive for M. ovipneumoniae. Viral cultures yielded an as yet unidentified herpesvirus. Sheep in the original and new herds had no serologic titers to parainfluenza-3, equine viral rhinopneumonitis, or infectious bovine rhinotracheitis, and had variable titers against bovine respiratory syncytial virus. No titers against M. ovipneumoniae were present in 13 sheep still in the original exhibit, but titers varied from 1:32 to 1:256 in eight pneumonic sheep. Sera taken from three sheep before or early in the outbreak were all negative for antibody to M. ovipneumoniae. Two of the affected Dall's sheep had been in contact with domestic sheep in the winter of 1985-1986, and M. ovipneumoniae was subsequently cultured from the domestic flock. Exposure to a new pathogen, and environmental and social stress in a new exhibit may have resulted in this severe disease in Dall's sheep.     

A preliminary report on the effect of dietary energy on prostaglandin F2 alpha production in vitro, interferon-tau synthesis by the conceptus, endometrial progesterone concentration on days 9 and 15 of pregnancy and associated rates of embryo wastage in ewes

Two groups of ewes were fed to provide 1.70 x (high energy group; n = 15) or 0.56 x (low energy group; n = 15) energy requirements for maintenance of liveweight from 14 d before a synchronized mating in November until slaughter at 9 or 15 d after mating. We investigated the effects on interferon-tau (IFN tau) secretion by the conceptuses, prostaglandin F2 alpha (PG) production in vitro by endometrial tissue, and associated rates of embryo mortality, endometrial progesterone content and progesterone production by luteal tissue. No differences between groups in pregnancy rate were detected on Day 9 between the 2 groups. Proportionately (6/6 vs 2/5), there were more pregnant ewes in the high energy group on Day 15, although this difference did not reach significance (P = 0.06). The proportion of corpora lutea represented by embryos was significantly lower in undernourished ewes (P < 0.05). Secretion in vitro of PG was lower in the 2 pregnant ewes of the low energy group on Day 15, and it was accompanied by higher IFN tau secretion by conceptuses recovered from these ewes. However, the limited number of pregnant ewes recorded on Day 15 prevented any statistical comparison. Neither mean endometrial content of progesterone nor ovarian venous progesterone concentrations and production of progesterone by luteal were affected by nutrition. The provisional results of the present experiment indicate that undernutrition may induce a reduction in the rate of secretion of IFN tau and can therefore increase production of PG from the endometrium. This could initiate luteolysis. The lower pregnancy rates observed in underfed ewes could be mediated through this alteration in the signal of maternal recognition of pregnancy. However, these findings remain to be shown in further experiments including a larger number of animals, as they only represent data from 2 undernourished animals.     

Production of identical twins by bisection of blastocysts in the cow

Day-8 embryos were recovered by a non-surgical method from superovulated crossbred heifers. Normal expanded blastocysts with a distinct inner cell mass and a trophoblast were released from the zona pellucida and bisected along a sagittal plane into two 'half' blastocysts. Each 'half' blastocyst was replaced in an empty zona pellucida and cultured for 2 h in B2 medium. After culture the 'half' blastocysts were directly transferred to recipient heifers via the cervix. From 11 blastocysts, 11 monozygotic 'half' blastocyst pairs were transferred to 11 recipients: 8 recipients became pregnant, 4 carried twins and one delivered a normal calf and an acardiacus amorphus monster consisting of disorganized embryonic tissues. A further 11 'half' blastocysts were transferred as singletons to 11 recipients. Five recipients were apparently pregnant at Day 42. One returned to oestrus at Day 45, 3 were carrying normal fetuses and 1 a pair of normal twin fetuses when slaughtered at Day 128. It is concluded that even after the first irreversible cellular differentiation which occurs at the blastocyst stage it is still possible to produce identical cattle twins by bisection of the Day-8 blastocyst.     

Diagnosis of pregnancy in the ewe at mid-gestation

Detection of the pregnancy-specific antigen, chorionic somatomammotrophin in serum was applied to the diagnosis of pregnancy in a commercial-type flock of 286 sheep 70 days after joining with rams. At the time of testing the ewes were between Days 47 and 70 of pregnancy. Based upon lambing results, the positive diagnoses were 97% correct. However, the accuracy in diagnosing non-pregnancy, which rose from 85% when all the ewes (Day 47–70) were considered to 99% after Day 55, suggest that some pregnant ewes whose stage of pregnancy was earlier than Day 55 were not being detected and were wrongly designated non-pregnant. This pregnancy test can be successfully used from Day 55, although a correct prediction of pregnancy was made in 40 ewes between Days 47 and 54.     

Ram novelty and the duration of ram exposure affects the distribution of mating in ewes exposed to rams during the transition into the breeding season

This study compared the affect of short-term and continuous exposure to rams during the transition between anoestrus and the breeding season on the distribution of mating and subsequent lambing. Further, within ewes continuously exposed to rams we investigated the effect of replacing these rams every 17 days with 'novel' rams. During August (late anoestrus, Northern Hemisphere), multiparous, North of England mule ewes were allocated to one of four groups: SVR ewes were exposed to vasectomised rams for 24h on Day 0 (short term; n=109), RVR ewes were exposed to vasectomised rams for 24h on Days 0, 17 and 34 (short term; n=113); PVR ewes were exposed to vasectomised rams on Day 0 and remained with the same rams for the duration of the pre-mating period (continuous; n=104); NVR ewes were continuously exposed to vasectomised rams from Day 0 with the rams replaced with 'novel' rams every 17 days (continuous; n=113). Blood samples were collected from a subset of ewes (n=22 per group) to monitor progesterone. On Day 50, harnessed, entire rams were introduced for mating and raddle marks recorded daily for the first 17 days. The median date of mating occurred 1 day earlier in NVR ewes than PVR ewes (P<0.05). A synchrony score calculated from the blood sampled ewes showed that the distribution of mating was more synchronised in PVR and NVR ewes than SVR and RVR ewes (P<0.001). PVR and NVR ewes had an earlier onset of cyclic activity than RVR ewes (P<0.01). However, only NVR ewes differed from SVR ewes in this variable (P<0.05). Within ewes lambing to first service, the median date of lambing of PVR, NVR and SVR ewes occurred at least 2 days earlier than RVR ewes (at least P<0.05). Further, PVR and NVR ewes had a more compact distribution of lambing than SVR and RVR ewes (P<0.05) and lambing was more compact in NVR ewes than PVR ewes (P<0.05). In conclusion, ewes in continuous contact with rams prior to mating had a more synchronised distribution of mating and lambing than ewes given only short-term exposure to rams. This distribution of mating in continuous ram exposed ewes can be further enhanced by periodic exposure to novel rams.