Investigation of factors affecting pregnancy rate after embryo transfer in the dromedary camel

The uteri of 36 adult dromedary camels were flushed non-surgically three times each with 90-120 mL of embryo flushing medium 7 days after ovulation. A total of 242 embryos were recovered, of which 139 were transferred non-surgically to recipient camels that were either at different levels of synchrony with respect to the Day 7 donor (+1 to -3 days; n = 58), or were at Day 6 after ovulation, but received one of the following treatments: (i) none (controls, n = 15); (ii) 150 mg progesterone-in-oil injected intramuscularly once daily during Days 5-20 after ovulation inclusive (n = 16); (iii) 500 mg flunixin meglumine given intravenously 15 min before transfer of the embryo (n = 6); (iv) 20 microg of the gonadotrophin-releasing hormone (GnRH) analogue buserelin given on Day 5 after ovulation (n = 12); or (v) the embryo was cooled to 4 degrees C and held at this temperature in an insulated container for 24 h before being transferred (n = 32). Jugular vein blood samples, taken daily from all the recipient camels during Days 0-20 after ovulation, were assayed for progesterone concentration and closely timed serial samples taken from the camels receiving flunixin meglumine or GnRH were assayed for 13,14-dihydro-15-keto prostaglandin F2alpha (PGFM) or oestradiol concentrations. The pregnancy rate increased to a maximum of 67% when ovulation in the recipient was negatively synchronized to have occurred 1 day behind that in the donor, and it fell dramatically when the level of asynchrony between recipient and donor increased to +1 (9%) or -3 (10%) days. It was not improved by daily injections of progesterone (44%), flunixin meglumine given before transfer (16%), or GnRH given on Day 5 (33%). Of the 32 embryos that were cooled to 4 degrees C before being transferred to Day 6 recipients, 20 resulted in pregnancies (63%) to give a success rate similar to that attained with the control fresh embryos (67%). Serum progesterone concentrations in the recipients increased to a mean +/- SEM of 2.6 +/- 0.8 ng mL(-1) by Day 8 after ovulation and, in those that were pregnant, levels remained elevated at 3-5 ng mL(-1) for the remainder of the sampling period; in non-pregnant recipients the concentrations declined to <1 ng mL(-1) by Day 11. Plasma PGFM concentrations in the flunixin meglumine-treated camels remained low (40-90 pg mL(-1)) compared with those in the untreated control camels, in which peak values of around 180 pg mL(-1) were reached within 10 min after transfer after which a steady decline occurred until resting concentrations of 90-100 pg mL(-1) were reached by 110 min after transfer. Treatment with GnRH on Day 5 after ovulation produced a transitory increase in serum oestradiol-17beta concentrations for 24 h. However, from Day 8, oestradiol concentrations in both the GnRH-treated and the untreated camels increased steadily to reach 2.5-3.5 pg mL(-1) by Day 12.     

Embryo transfer in the dromedary camel (Camelus dromedarius) using non-ovulated and ovulated, asynchronous progesterone-treated recipients

The aim of the present study was to investigate the use of exogenous progesterone and equine chorionic gonadotrophin (eCG) in non-ovulated and ovulated, asynchronous dromedary camel recipients being prepared for an embryo transfer programme. The uteri of 12 mated donor camels were flushed non-surgically 7 days after ovulation and 42 embryos were recovered. In Experiment 1, 16 embryos were transferred non-surgically to recipients on Day 3 or 4 after ovulation (ov+3 and ov+4, respectively). Each recipient received a daily dose of 75 mg, i.m., progesterone-in-oil from 2 days before embryo transfer until 6 days after ovulation. Thereafter, the progesterone dose was reduced to 50 mg on Day 7 and finally to 25 mg day(-1) on Days 8 and 9. Nine of 16 recipients (56%; ov+3, n=4; ov+4, n=5) became pregnant compared with none of eight non-progesterone treated controls, into which embryos were transferred on Day 4 after ovulation. In Experiment 2, 18 non-ovulated recipients received 75 mg, i.m., progesterone-in-oil daily from 3 days before until 12 days after non-surgical transfer of a Day 7 blastocyst, at which time pregnancy was diagnosed by ultrasonography. All pregnant recipients continued to receive 75 mg progesterone-in-oil daily for a further 6 days, when each camel received 2000 IU, i.m., eCG. Progesterone treatment was then reduced to 50 mg day(-1) and, when a follicle(s) ≥1.3 cm in diameter were present in the ovaries, each animal received 20 μg buserelin to induce ovulation. Once the corpora lutea had developed, progesterone treatment was reduced to 25 mg day(-1) for a final 3 days. Fourteen of 18 recipients (78%) became pregnant and seven of these (50%) remained pregnant after eCG treatment. Of the seven pregnancies that were lost, two were lost before eCG treatment, two did not respond to eCG treatment and three responded to eCG treatment and ovulated, but lost their pregnancies 6-8 days after the last progesterone injection.     

Comparison of two different methods for the vitrification of hatched blastocysts from the dromedary camel (Camelus dromedarius)

The uteri of 32 donor camels were flushed non-surgically on Day 6, 7 or 8 after ovulation and a total of 184 embryos was recovered. Sixty Day 6 embryos and 61 Day 7 embryos were vitrified or frozen ultrarapidly using open pulled straws and a modified version of the Vajta protocol. These embryos were subjected to concentrations of either 10% and 20% or 20% and 40% ethanediol as the cryoprotectant before being loaded into open pulled straws (OPS) and plunged into liquid nitrogen. All embryos were subsequently thawed and rehydrated either directly into holding media or into holding media containing 0.2 M sucrose and were incubated for 5 or 10 min before being transferred to holding media before transfer to recipients. Although the survival rate of the embryos immediately after thawing was high (OPS 20%/40% ethanediol resulted in 97% and 100% survival for Day 6 and Day 7 embryos, respectively; OPS 10%/20% ethanediol resulted in 90% and 70% survival for Day 6 and Day 7 embryos, respectively), after 2 h in culture, survival rates had decreased to 46% and 53% for Day 6 and Day 7 embryos, respectively, using OPS 10%/20% and 53% and 63% for Day 6 and Day 7 embryos, respectively, using OPS 20%/40%; however, none of the embryos transferred resulted in a viable fetus. A further 63 embryos (Day 6: n = 31; Day 7: n = 16; Day 8: n = 16) were subsequently exposed to vitrification solution (20% glycerol + 20% ethylene glycol + 0.3 M sucrose + 0.375 M glucose + 3% polyethylene glycol) in three steps and after loading into 0.25 mL straws were plunged into liquid nitrogen. However, a much greater percentage of the Day 7 and Day 8 embryos (43.8% and 81.2% respectively) were fractured or torn after warming and none of the 12 intact embryos transferred resulted in a pregnancy. Better survival rates immediately after thawing and rehydration were obtained with the smaller Day 6 embryos (94%), which resulted in a total of eight fetuses from the 21 embryos transferred.     

Effect of embryo source and recipient progesterone environment on embryo development in cattle

The aim of the present study was to examine the effect of embryo source (in vivo v. in vitro) and the progesterone environment into which it was transferred on Day 7 on embryo survival and size on Day 13. Day 7 blastocysts were produced either in vivo using superovulation, artificial insemination and non-surgical embryo recovery or in vitro using in vitro maturation, fertilisation and culture. In order to produce animals with divergent progesterone concentrations, following synchronisation recipients were either superovulated (High progesterone; n = 10) or not (Control progesterone; n = 10). Ten blastocysts, produced either in vivo or in vitro, were transferred to each recipient on Day 7. Both groups were killed on Day 13. The mean progesterone concentration from Day 7 to Day 13 (the period when the embryos were in the uterus) in the High and Control progesterone recipients was 36.32 +/- 1.28 and 10.30 +/- 0.51 ng mL(-1), respectively. Of the in vivo embryos transferred, the overall recovery rate at Day 13 was 64%, which was higher (P < 0.001) than that of 20% for the in vitro embryos transferred. The mean area of embryos recovered from High progesterone recipients was 3.86 +/- 0.45 mm(2) (n = 28) compared with 1.66 +/- 0.38 mm(2) (n = 24) for embryos recovered from Control progesterone recipients (P < 0.001). Similarly, the origin of the embryo used for transfer affected embryo size on Day 13. In summary, the recovery rate of blastocysts was higher for in vivo- than in vitro-derived embryos. Blastocyst size was approximately 2.3-fold greater in recipients with high compared with normal progesterone. The present study lends strong support to the hypothesis that an earlier rise in progesterone after conception stimulates blastocyst growth and the development of competent embryos.     

Developmental competence in vitro and in vivo of cryopreserved, hatched blastocysts from the dromedary camel (Camelus dromedarius)

The present paper describes experiments designed to investigate methods for cryopreserving embryos from dromedary camels. Because preliminary studies had shown ethanediol to be the best cryoprotectant to use for camel embryos, the current experiments were performed to determine the minimum exposure time to 1.5 m ethanediol required to achieve cryoprotection. The uteri of 30 donor camels were flushed non-surgically 8 days after mating. Embryos were recovered and 158 were assigned to one of three groups, which were exposed to 1.5 m ethanediol for either 10 min (n = 67), 5 min (n = 51) or 1 min (n = 40). Embryos were subsequently thawed and rehydrated by expelling either directly into holding medium (HM; HEPES-buffered Tyrode's medium containing sodium lactate and 3 mg mL(-1) bovine serum albumin, 10% fetal calf serum, 100 IU mL(-1) penicillin G, 100 microg mL(-1) streptomycin and 25 microg mL(-1) amphotercin B) or initially into HM containing 0.2 m sucrose for 5 or 10 min. The survival rate of all embryos immediately post-thawing, as judged by the morphological appearance of the embryos, was high (91%), but was greatly reduced after 2 h culture (59%). Ninety-two embryos were transferred to recipient camels resulting in 18 viable fetuses (1 min ethanediol exposure, n = 1/15; 5 min ethanediol exposure, n = 3/34; 10 min ethanediol exposure, n = 14/43). Of the embryos rehydrated directly in HM, six of 65 resulted in viable fetuses and those rehydrated initially in 0.2 m sucrose for 5 or 10 min resulted in nine of 47 and three of 46 fetuses respectively. From these experiments, we conclude that camel embryos can be cryopreserved using ethanediol as a cryoprotectant when the embryos are cooled slowly (to 33 degrees C) before being plunged into liquid nitrogen for storage.     

Assisted reproductive techniques for hybridization of camelids

The camelid family comprises the Old World camelids (or dromedary and Bactrian camels) and the New World camelids (namely the llamas, alpacas, guanacos and vicunas). Although the species within each group can hybridize among themselves to produce fertile offspring, it is only recently that a hybrid between New and Old World camelids has been reported. To create this hybrid, semen was collected from male camels by artificial vagina (AV) and inseminated into female guanacos (n = 9) and llamas (n = 3) at the appropriate stage of their follicular wave cycle. Similarly, guanaco and llama semen was collected, also by AV, and inseminated into female camels (n = 42). Although several conceptions occurred, only one hybrid (camel sire x guanaco dam) continued to term and was born alive after 328 days of gestation, and another is pregnant at the time of writing (camel sire x llama dam). Further studies are presently being carried out using extraspecific embryo transfer to try and improve the success rate of live offspring being born. Female guanacos (n = 4) are treated with hormones to stimulate their ovaries to produce several follicles before being inseminated with camel semen. Of the 12 camel recipients that have to date received hybrid embryos (camel sire x guanaco dam), 10 conceived, but 9 of these subsequently aborted between 30 and 365 days and only one recipient was still pregnant at the time of writing.     

Pulsatile release of luteinising hormone during the luteal phase in lactating and weaned sows

The present paper describes luteal phase luteinising hormone (LH) in sows that ovulated due to a limited nursing regimen (LN). The LN regimen was imposed either at Day 14 or at Day 21 of lactation. At ovulation, lactation was terminated (n = 8) or sows remained lactating throughout early pregnancy (n = 8). Blood samples were collected every 15 min for 8 h during the day, around Day 11 after ovulation. In addition, lactating sows were bled during the night, when piglets were allowed to suckle. The LH pattern was typical for the luteal phase, with one to five pulses per 8 h. The LH characteristics (frequency, base, average, pulse area) did not differ between lactating and weaned sows, except for the amplitude of LH pulses, which was higher in weaned sows compared with lactating sows (1.22 +/- 0.15 v. 0.76 +/- 0.11 ng mL(-1), respectively; P < 0.05). In lactating sows, average LH, basal LH and the frequency of LH pulses were significantly lower during the night, when piglets were allowed to suckle. The sage of lactation at which LN was imposed did not influence LH. In conclusion, it is unlikely that the small difference in LH explains the considerable difference between lactating and weaned sows in progesterone at Day 12 of pregnancy (24.1 +/- 1.3 v. 43.3 +/- 4.0 ng mL(-1), respectively; P < 0.01). Moreover, the difference in progesterone already exists during the early luteal phase (Day 0-10), when secretion of the corpora lutea is still independent of LH.     

The viability of transferred sheep embryos after long-term cryopreservation

The survival to term of 414 sheep embryos, thawed and transferred after conventional cryopreservation and storage for 13 years, was evaluated. A concurrent experiment involving the transfer of vitrified embryos to 91 ewes and artificial insemination of 51 ewes with frozen-thawed semen from sires of the long-term cryopreserved embryos provided forms of control treatments. The donor ewes had a mean ovulation rate of 10.9, and 7.1 embryos per ewe were cryopreserved. Each recipient ewe received two embryos and pregnancy was assessed at Day 18, Day 54 and term. The pregnancy rate was lower in the long-term embryo group than the artificial insemination group at Day 18 (P<0.01) and Day 54 (P<0.05), although the difference at term (31% v. 49%) was not significant, with the vitrified embryo group being similar to the long-term group. Embryo survival to birth was 21%, with the majority of loss (80%) occurring by Day 18. The later stage of development and higher grade of transferred embryos and the older age of donor ewes resulted in a significantly higher (P<0.01) pregnancy rate at Day 54 and term, and percentage of lambs born and weaned. Other effects of donor ewes (genotype, superovulation treatment, number of ovulations and embryos cryopreserved) were not significant. Implications for the design of genetic evaluation and germplasm conservation programmes using embryo cryopreservation technology are discussed.     

Cloned horse pregnancies produced using adult cumulus cells

The objectives of the present study were to: (1) clone horses using adult cumulus cells; and (2) determine whether the cumulus cell donor affected the outcome. In vivo-matured cumulus-oocyte complexes were obtained using transvaginal ultrasound-guided follicle aspiration; oocytes were used as cytoplasts, whereas cumulus cells (from one of three different mares) were used as donor cells. Immediately following nuclear transfer and activation procedures, cloned embryos were transferred surgically to the oviduct of recipient mares (n = 2-5 embryos per recipient) that had ovulated within 24 h prior to the transfer. An initial pregnancy examination was performed between Days 14 and 16 (Day 0 = surgery); subsequent examinations were then performed every 7-10 days. A total of 136 follicles were aspirated in 96 mares, from which 72 oocytes were recovered (53%). Sixty-two cloned embryos were transferred to recipient mares, which resulted in seven (11.3%) ultrasonographically detectable conceptuses between Days 14 and 16. All seven conceptuses were lost spontaneously between Days 16 and 80. Cumulus cells from Mare 160 tended (P = 0.08) to result in a higher embryo survival rate than cumulus cells from Mare 221 (4/17 v. 1/25 respectively). To our knowledge, this is the first report documenting the establishment of cloned equine pregnancies derived from adult cumulus cells.     

Embryonic disc development and subsequent viability of cattle embryos following culture in two media under two oxygen concentrations

Bovine embryos were produced in vitro using a 2 x 2 design of modified medium (KSOM or SOF) and oxygen concentration (5% or 20%). Day 7 blastocysts were transferred in bulk (n = 11, on average) to recipient heifers and recovered non-surgically at Day 14. In two replications of a Latin square, eight heifers received embryos from each combination of factors. Recovered embryos were evaluated for trophoblast length and width, as well as the presence and diameter of an embryonic disc (ED). An ED was detected in a higher percentage of embryos that had been cultured in KSOM than SOF (72% v. 46%, respectively; P < 0.05). The aim of a second series of experiments was to associate Day 14 morphology with subsequent developmental capacity. In vitro-produced blastocysts were transferred (n = 17-20) on Day 7 to each of eight heifers and recovered at Day 14. Thirty-eight blastocysts were retransferred to heifers following morphological evaluation. Embryos in which an ED with no signs of degeneration had been detected maintained more pregnancies than other embryos in which an ED had either shown signs of degeneration or had not been detected (5/8 v. 2/30, respectively; P < 0.01). Further investigation into ED integrity at the elongating stage may contribute to our understanding of pregnancy establishment and maintenance.     

白细胞介素-1β,-1Rα对胚胎-内膜共培养体系分泌基质金属蛋白酶-9/细胞间黏附分子-1的影响

目的 探讨白细胞介素(IL)-1β,-1Rα在胚胎-内膜共培养体系中的作用.方法 选取昆明种雌性小白鼠30只(6～8周龄),雄性15只(12～16周龄)进行实验.30只雌鼠成功妊娠后,收集雌鼠8细胞期胚胎并进行子宫内膜腺上皮细胞原代培养.共培养成功7份原代子宫内膜细胞,每份细胞分别接种35孔(n=35×7).5孔细胞培养液中不做任何处理(对照组,n=5×7),其余30孔根据上皮细胞培养液中所含IL-1β浓度不同以及IL-1β+IL-1Rα比例不同共分为6组,分别为1 ng/mL IL1β组,10 ng/mL IL-1β组和100 ng/mL IL 1β组,以及1 ng/mL IL-1β+10 μg/mL IL-1Rα组,10 ng/mL IL 1β+10 μg/mL IL-1Rα组,100 ng/mL IL-1β+10 μg/mL IL-1Rα组(每组样本量:n=5×7).将发育正常的8细胞胚胎随机置人含以上不同培养液的细胞孔内,每孔6枚胚胎,培养48 h后收集培养上清液,采用酶联免疫吸附测定(ELISA)法检测培养液中分泌蛋白基质金属蛋白酶(MMP)-9/细胞间黏附分子(ICAM)-1的表达水平.结果 1 ng/mL IL-1β组与10 ng/mL IL-1β组,可显著促进共培养体系MMP-9/ICAM-1的表达,与对照组比较,差异有统计学意义(P＜0.05);而1 ng/mL IL-1β组与10 ng/mL IL-1β组,以及100 ng/mL IL-1β组分别与对照组比较,差异均无统计学意义(P＞0.05).10 ng/mL IL-1β+ 10 μg/mL IL 1Rα组MMP-9/ICAM-1水平显著低于对照组(P＜0.05);而1 ng/mL IL-1β+ 10 μg/mL IL-1Rα组,100 ng/mLIL-1β+10 μg/mL IL 1Rα组分别与对照组比较,差异均无统计学意义(P＞0.05);1 ng/mL IL-1β+ 10 μg/mLIL-1Rα组与100 ng/mL IL-1β+10 μg/mL 1L-1Rα组比较,差异亦无统计学意义(P＞0.05).结论 IL-1β/IL-1Rα的适当比例,可以调控胚胎着床.     

In vitro embryo culture in the production of identical merino lambs by nuclear transplantation

This study examined the viability of embryos developed in vitro from 8- to 16-cell stage blastomeres fused with enucleated oocytes. Of 209 blastomeres recovered and subjected to manipulation and electrofusion procedures, 190 (91%) fused successfully, with 86 (45%) of those undergoing cleavage up to the 4- to 16-cell stage when cultured for 66 h in a synthetic oviduct fluid medium. The viability of the embryos was examined by transferring them to recipient ewes and determining the ewes' pregnancy status by ultrasound on Day 45. Of 86 embryos transferred, 14 developed to fetuses in 8 of the 36 recipients, including four sets of identical twins and one set of quads. In contrast, with uncultured and unmanipulated embryos, 15 fetuses developed from 19 embryos transferred at a similar stage of development. The viability of embryos derived from manipulated zygotes cultured in vitro was comparable to that previously reported for studies employing in vivo culture, indicating the potential of in vitro culture systems based on a simple medium for nuclear-transplantation embryos.     

In vitro assessment of the viability of sheep zygotes after pronuclear microinjection

Microinjected sheep zygotes were cultured in synthetic oviduct fluid medium (SOFM) for either 1 or 3 days and their subsequent developmental capacity was compared with that of microinjected zygotes cultured in vivo. Two experiments were carried out, using zygotes microinjected with one of three gene constructs containing the CysE and CysM genes from Salmonella typhimurium. In Experiment 1, microinjected zygotes were allocated to one of three treatments: (1) immediate transfer to recipient ewes (in vivo culture) followed by recollection 1 or 3 days later and subsequent transfer of viable embryos to other recipient ewes, (2) culture in SOFM (in vitro culture) for either 1 or 3 days before transfer to recipient ewes, and (3) immediate transfer to recipient ewes without subsequent interference. Recipient ewes were slaughtered on Day 14 of pregnancy and the number of elongated conceptuses determined. Although fewer zygotes failed to divide during in vitro culture than during in vivo culture, there were, overall, no significant differences between treatments in the percentage of zygotes that developed into elongated conceptuses (32.6-50.0%). In Experiment 2, microinjected zygotes were transferred immediately to recipient ewes or cultured in vitro for either 1 or 3 days before transfer. The number of fetuses per ewe on Day 50 of pregnancy and the number of lambs delivered per ewe were recorded. Neither the percentage of recipient ewes that became pregnant (overall 114/166, 68.7%) nor the percentage of zygotes that developed into lambs (overall 186/803, 23.2%) was significantly influenced by the culture treatment or by the gene construct microinjected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of luteectomy on the maintenance of pregnancy, circulating progesterone concentrations and lambing performance in sheep

In sheep, there have been few and conflicting data regarding the necessity of the corpus luteum (CL) for the maintenance of pregnancy. The aims of the present study were to examine the effect of luteectomy on and after Day 50 of pregnancy on maternal plasma progesterone concentrations and the progression of pregnancy, to determine the minimum placental progesterone support required for the maintenance of pregnancy, and to evaluate the effect of luteectomy on lambing performance. In Experiment 1, four ewes luteectomized on Day 50 of pregnancy aborted 2-7 days after surgery, whereas pregnancy progressed and parturition occurred between Days 143 and 149, with live lambs, in three of four ewes and in four ewes luteectomized on Days 60 and 70 of pregnancy respectively. The mean (+/- SEM) progesterone concentrations on the day before and one day after luteectomy decreased from 4.87+/-0.85 to 0.42+/-0.06 ng mL(-1) (P<0.01), from 4.57+/-0.51 to 0.80+/-0.12 ng mL(-1) (P<0.02) and from 6.05+/-0.52 to 1.67+/-0.11 ng mL(-1) (P<0.01), respectively, for the ewes luteectomized on Days 50, 60 and 70 of pregnancy. The fall in progesterone concentrations was 90%, 80% and 71%, respectively, for the ewes luteectomized on Days 50, 60 and 70 of pregnancy. In Experiment 2, pregnancy progressed in four ewes luteectomized on Day 70 and parturition occurred between Days 146 and 149, with live lambs. The mean progesterone concentrations declined (P<0.01) from 6.9+/-0.7 ng mL(-1) on the day before luteectomy to 2.1 = 0.3 ng mL(-1) the day after surgery. The concentrations of progesterone in blood collected every 3 h during a 24-h period were stable on Days 60 and 80 of pregnancy, but they were lower (P<0.03) on Day 80 than on Day 60 of pregnancy, for each time period examined. In Experiment 3, the gestation length and birthweights of single, twin and triplet lambs were not different between the control intact ewes (n = 111) and the ewes luteectomized on Days 70-80 of pregnancy (n = 71). Lamb mortality was not different between the two groups (7.2% v. 8.4%, control v. luteectomized). In conclusion, these results showed that (1) the sheep CL is necessary to maintain pregnancy until at least Day 60, (2) progesterone withdrawal induced by luteectomy on and after Day 50 of pregnancy must be of a critical magnitude to provoke abortion, (3) after Day 60 of pregnancy, the CL and the placenta together secrete more progesterone than required for pregnancy maintenance, (4) there is no apparent 24-hour rhythm in maternal plasma progesterone concentrations before and after luteectomy, and (5) luteectomy at mid pregnancy has no apparent effect on gestation length, lamb birthweight or lamb mortality.     

Effects of cobalt/vitamin B12 status in ewes on ovum development and lamb viability at birth

Scottish Blackface ewes from cobalt-deficient farmland were fed a diet containing 0.06 mg cobalt per kg dry matter from approximately 30 days before embryo recovery/transfer until lambing. Ewes remained untreated (-Co; n = 82) or were given an intraruminal cobalt-containing bolus to compensate for the dietary deficit (+Co; n = 82). Ewes used as embryo donors (-Co, n = 17; +Co, n = 16) were artificially inseminated with semen from a single Suffolk sire. Day 6 embryos obtained from -Co and +Co donors were transferred in singleton to -Co and +Co recipients in a 2 x 2 factorial-designed experiment to determine the effects of cobalt/vitamin B12 status during the periconception period (factor 1) and pregnancy (factor 2) on lamb viability at birth. Mean (+/- s.e.m.) circulating concentrations of vitamin B12 in -Co and +Co donors at ovum recovery were 182 +/- 10 and 1288 +/- 64 pmol L(-1), respectively (P < 0.001), and the number of corpora lutea per ewe ovulating was 9.9 +/- 1.6 and 14.4 +/- 1.3, respectively (P < 0.05). Treatment did not affect the proportion of recovered ova that contained >32 cells (viable) or the median stage of development (late morula), but viable ova recovered from -Co v. +Co ewes had a better morphological grade (2.0 +/- 0.1 v. 2.20 +/- 0.04, respectively; P < 0.01). There was no effect of treatment on the proportion of recipient ewes that became pregnant. Circulating concentrations of vitamin B12 were lower in -Co than +Co ewes during pregnancy (P < 0.001) and at birth in lambs born to -Co ewes compared with those born to +Co ewes (P < 0.001). There was no effect of donor or recipient cobalt/vitamin B12 status on lamb birthweight, neonatal vigour or neonatal rectal temperatures, but lambs derived from +Co v. -Co embryo donors were more active in the first 3 days after birth (P < 0.05). Results show that sub-clinical cobalt/vitamin B12 deficiency reduces ovulatory response in superovulated ewes and that periconception nutrition can affect neonatal lamb behaviour.     

Fetal growth and development following temporary exposure of day 3 ovine embryos to an advanced uterine environment

The effect of exposing Day 3 ovine embryos to an advanced uterine environment for a period of 3 days on subsequent fetal growth and development between Day 35 and Day 135 of gestation was studied. Day 3 embryos were recovered from superovulated donor ewes and transferred to synchronous final or asynchronous temporary recipients for 3 days. Embryos were recovered from these temporary recipients and transferred to Day 6 final recipients. Gravid uteri were recovered, weighed and dissected on Days 35, 45, 60, 90, 110, 125 and 135 of gestation. Fetal weight and length data were analysed by fitting non-linear Gompertz models of the form log(e) y = a - be(-ct), where y is fetal size and t is time from conception. Various terms including treatment, gestational age, embryo stage at transfer and fetal sex were fitted to this model. Fetal development was assessed by relating organ weight to fetal bodyweight using the linear allometric equation log(e) y = log(e) a + b log(e) x, where y is organ weight and x is fetal weight. Temporary exposure of Day 3 embryos to an advanced uterine environment did not increase the rate of embryo development and had no effect on fetal growth and development between Days 35 and 135 of gestation in this study. A single Gompertz model (log(e) y = 10.134 - 17.047e(-0.1733t)) explained 99.8% of the variation in fetal weight. Of terms fitted to this model only gestational age and fetal sex influenced fetal weight, with male fetuses being 5% heavier (P<0.05) than female fetuses. Fetal development was also unaffected by experimental treatment in this study. Allometric coefficients established for various fetal components agreed well with those from previously published studies.     

Lunelle monthly contraceptive injection (medroxyprogesterone acetate and estradiol cypionate injectable suspension): assessment of return of ovulation after three monthly injections in surgically sterile women

The pharmacodynamic effects of medroxyprogesterone acetate (MPA) and estradiol cypionate (E2C) (MPA/E2C) (Lunelle Monthly Contraceptive Injection) on ovarian function were assessed through changes in serum progesterone concentrations. The data described here were obtained simultaneously with pharmacokinetic data presented in another article in this issue. Sixteen surgically sterile women with regular menstrual cycles were studied for one control cycle, three consecutive treatment months, and 3-5 months of follow-up. Suppression, followed by resumption of ovulation (the dynamic end point), was assessed by serum progesterone levels. Return of ovulation was presumptive based on progesterone concentrations > or = 4.7 ng/mL, as ultrasound was not used to determine the follicular/ovulatory status of these subjects. Luteal-like serum progesterone peaks were observed in all 16 women before drug administration, confirming the presence of ovulatory cycles. After the third monthly injection of MPA/E2C, progesterone concentrations were measured until demonstration of ovulation. Two women discontinued and three were lost to follow-up before this objective was achieved. Serum progesterone levels and, consequently, ovulation were suppressed beyond the entire dosing interval, indicated by the absence of any luteal-like progesterone peaks (serum progesterone concentrations did not exceed 1 ng/mL). The first normal ovulatory cycle, based on progesterone concentrations > or = 4.7 ng/mL, was observed in 11 women between days 63 and 112 after the third injection. Select medroxyprogesterone acetate parameters (i.e., area under the curve and minimum concentration) were correlated with return of ovulation. The correlation coefficients (r) were 0.757 and 0.492 for area under the curve and minimum concentration, respectively, indicating that return of ovulation is dependent, in part, on area under the curve and on the magnitude of the serum MPA trough level. In general, the higher the minimum concentration levels, the longer the time to return of ovulation. In conclusion, the return of ovulation, as confirmed by serum progesterone concentrations > or = 4.7 ng/mL, was observed as early as 63 days after the third and final monthly intramuscular injection of MPA/E2C, suggesting that consistent suppression of the hypothalamic-pituitary-ovarian axis is reversible after discontinuation of dosing.     

Luteal stage dependence of pituitary response to gonadotrophin-releasing hormone in cyclic dairy ewes subjected to synchronisation of ovulation

Possible hormonal aberrations precluding conception or maintenance of pregnancy in dairy ewes subjected to ovulation synchronisation were investigated in this study. The pituitary response to exogenous gonadotrophin-releasing hormone (GnRH) was tested at different luteal stages in 36 ewes. Oestruses were synchronised by using progestagen-impregnated sponges and the animals were randomly allotted into one of three treatment groups (A, B and C; n = 12 for each). Treatments commenced on Days 4, 9 and 14 of the new cycle (oestrus was defined as Day 0). Ewes were given two GnRH injections, 5 days before and 36 h after a prostaglandin F2+/- (PGF2+/-) injection, and the animals were inseminated 12-14 h after the second GnRH injection (modified OVSYNCH). For luteinising hormone (LH) determination blood samples were withdrawn from six ewes of each group at the time of GnRH administration, and 30, 90, 180, 270 and 360 min later. Progesterone was assayed in samples taken every other day starting from oestrus and for 17 days after the second GnRH injection, and in an additional sample collected on the day of insemination. After the first GnRH injection, the LH concentration was higher in Group C than in Groups B and A (mean +/- s.d.: 64.8 +/- 10.0 ng mL(-1), 41.3 +/- 3.7 ng mL(-1) and 24.6 +/- 9.0 ng mL(-1), respectively; P < 0.05), whereas after the second GnRH injection a uniform LH release was found in all groups. PGF2+/- caused a significant decrease in progesterone (P4) concentration in all groups; however, at artificial insemination ewes that conceived had significantly lower P4 concentration in comparison with those that failed to conceive. As early as Day 5, pregnant animals had higher P4 concentrations than non-pregnant animals. Overall, 21 animals conceived (seven, nine and five ewes from Groups A, B and C, respectively). These results indicate that the proposed protocol is equally effective in inducing a preovulatory LH surge at any stage of the luteal phase, and that elevated P4 concentration along with a delayed P4 increase should be considered as a causative factor for inability to conceive.     

Reproductive cycles of farmed female chital deer (Axis axis)

Studies were conducted on chital deer hinds (Axis axis) living in a temperate region to advance the understanding of the patterns of reproduction of a tropical cervid species. The hinds exhibited regular patterns of oestrus cyclicity throughout the year as evidenced by concentrations of serum progesterone monitored over a 14-month period, and detection of behavioural oestrus by vasectomized stags. The mean length of the oestrous cycle was 18.0+/-0.7 days (range, 12-23 days). Profiles of serum progesterone showed concentrations of <0.5 ng mL(-1) at the time of oestrus, which rose to a peak (range 1.5-5.0 ng mL(-1)) about Day 13, and then declined to low concentrations at the next oestrus. Observations following parturition showed that the first detected oestrus occurred at a mean (+/- SEM) time of 26.9+/-3.0 days later for seven of nine hinds. The mean length of the oestrous cycle after the first post-partum oestrus was 16.6+/-1.0 days (range 7-20 days). The presence of a stag may influence the length of the post-partum period in chital deer hinds, and hinds in contact with a stag in this study had a significantly shorter interval from parturition to first ovulation (P<0.01) compared with hinds not in contact with a stag. By 7 weeks post partum a corpus luteum was detected in 93% of hinds. In comparison only 43% of hinds with no stag contact had a corpus luteum by 7 weeks post partum. It is suggested that the tendency towards seasonal calving in the study population may be related more to male than female factors.     

Effect of dietary intake on steroid feedback on release of luteinizing hormone in ovariectomized cows

This study tested the hypothesis that the decline in pulsatile release of luteinizing hormone (LH), resulting from steroid negative feedback, is greater in animals fed a low, compared with a high, plane of nutrition. Two-year-old cows were ovariectomized and six days later were fed diets to provide 1.5 x maintenance requirements (n = 6, supplemented) or 0.5 x maintenance requirements (n = 6, restricted) (Round 1). Pulsatile release of LH was measured over a 14-h period on the fifth day of feeding these diets (Day 1); at 6 h, all animals were treated with an intravaginal insert containing 1.38 g progesterone, which remained in place until the end of Day 3. Pulsatile release of LH was again measured for 14 h on Day 3; at 6 h, all animals were injected intramuscularly with oestradiol benzoate (ODB; 1 mg per 500 kg live weight). Three days later, this protocol was repeated, in a cross-over design, with cows that were previously restricted now being supplemented and those cows previously supplemented, now restricted (Round 2). Plasma concentrations of progesterone after intravaginal progesterone treatment were 1.01 ng mL(-1) higher in restricted cows compared with supplemented cows (P < 0.001) and were also higher in Round 1 than in Round 2 and on Day 1 than on Day 3 (P < 0.001). Plasma concentrations of oestradiol following injection with ODB did not differ between supplemented and restricted cows (P > 0.1). Dietary intake did not affect mean concentrations of LH, pulse frequency or amplitude during the 6-h period before steroid treatment or the change in these variables following steroid treatment; however, the slope of the decline in concentrations of LH following progesterone treatment was significantly more negative in cows fed restricted diets compared with those fed supplemented diets. In Round 2, mean concentrations of LH were higher preceding, and decreased more following, progesterone treatment compared with the decrease after ODB treatment. In conclusion, acute dietary restriction resulted in a more rapid decline in the release of LH following treatment with intravaginal progesterone, and was associated with higher concentrations of progesterone in plasma.