The effects of Trypanosoma congolense infection were investigated at the pituitary level on trypanosome resistant Baoulé bulls (aged 3–6 years), using immunohistochemistry of LH-and FSH-secreting cells and a combined dexamethasone and GnRH challenge. The pituitaries of two control and five naturally infected Baoulé bulls were removed after slaughter and the LH- and FSH-secreting cells were examined immunohistochemically, using specific polyclonal antibodies against βLH and βFSH. No significant impairment of the labelling and distribution of LH- and FSH-secreting cells was seen in infected bulls when compared with control animals. No parasites were found in the pituitary glands. Plasma LH and testosterone concentrations were determined in eight control and eight infected bulls by enzymeimmunoassay and radioimmunoassay techniques, respectively. Blood samples were collected at intervals of 30 min two times before and nine times after dexamethasone treatment (20 mg i.m.). GnRH (Busereline: 20 μg, i.m.) was injected 4.5 h later and samples were collected every 15 min for 180 min. After dexamethasone treatment, LH and testosterone concentrations declined dramatically in both groups. Four hours after treatment, the mean testosterone concentration for both groups was 0.44 ng ml−1. After GnRH injection, LH concentrations in the infected group increased rapidly to a mean maximum value of 30 ng ml−1 by 165 min. In contrast, the increase in LH concentration in non-infected bulls was more gradual and the mean maximum value, reached at the same time, was only 20 ng ml−1. Testosterone concentration increased rapidly and in a similar manner in both groups for the first 90 min (0.08 ± 0.04 ng ml−1). There was almost no further increase in testosterone concentration in the infected group (different from controls; P < 0.05) although LH concentrations continued to rise. The testosterone concentration of the non-infected group increased steadily, up to the end of the sampling period. It is concluded from the immunohistochemical study and from the pituitary response to GnRH that the parasites do not alter pituitary function but that they do affect testicular function.
D. Le Bourhis, P. Chesne, M. Nibart, J. Marchal, P. Humblot, J. P. Renard and Y. Heyman
The objectives of this study were to evaluate the efficiency and reliability of embryo sexing from isolated single blastomeres, and after nuclear transfer to examine the influence of the sex of donor embryos on development in vitro and in vivo up to calving. The sex of the donor embryo was determined by revealing a specific Y DNA sequence by PCR and electrophoresis after isolation of one, two, three, or more than five cells. The efficiency of sex determination was over 90% and reliability was 100% independent of the number of blastomeres used. In a second experiment, sex was determined from a single cell and the other cells were used for nuclear transfer. The effect of sex on in vitro development was studied in 386 male and 314 female reconstructed embryos derived from 19 male and 14 female parent embryos, respectively. Developmental competence in vitro of male and female constructs over 7 days was not statistically different (25.2 and 23.1% blastocysts on day 7, respectively; P > 0.05). After the transfer of predetermined male (n = 30) and female (n = 27) cloned embryos into recipient heifers, no effect of sex was observed on pregnancy rates at day 21, 35 and 90, or on calving rates (P > 0.05). These rates did not differ between single and twin transfer (P > 0.05). The sex of the calves born always corresponded to that determined from a single blastomere. These results show that sex can be determined accurately when using a single blastomere before nuclear transfer and that the sex of the parent embryo does not affect in vitro development or in vivo survival rates of cloned embryos.
P. Lonergan, H. Khatir, F. Piumi, D. Rieger, P. Humblot and M. P. Boland
In vitro produced bovine zygotes show substantial variation in the time required to complete the first cell cycle and in their in vitro development potential. A number of reports have highlighted the fact that the fastest developing embryos in vitro are most likely to be comparable with their in vivo counterparts. At 24 h after IVF, presumptive zygotes were cultured in droplets of synthetic oviduct fluid medium. Droplets were examined at regular intervals and all cleaved embryos at each time point were transferred into new droplets and cultured separately for the duration of the experiment. All uncleaved zygotes were returned to the incubator and re-examined at the successive time points until 48 h after insemination, at which time the remaining uncleaved oocytes were retained as a group. A representative number of day 7 blastocysts from zygotes that had cleaved by 30 or 36 h were transferred to synchronized recipients and pregnancy was diagnosed by ultrasonography at day 35. Glucose and glutamine metabolism was examined in zygotes and blastocysts and compared retrospectively with time of first cleavage. A representative number of blastocysts from each of the cleavage groups was sexed using PCR. Data were analysed by chi-squared and regression analysis. Development to the blastocyst stage decreased as the time from insemination to first cleavage increased (r = 0.97, P < 0.03). There was no difference in blastocyst hatching, number of blastocyst cells or pregnancy rate between the 30 and 36 h groups. The overall sex ratio was 62% males (n = 258, P < 0.0001) and was not different in the 30 and 36 h groups (61%, n = 155 versus 63%, n = 95, respectively). These results indicate that although time of first cleavage has a major influence on the probability of an embryo developing to the blastocyst stage, once that stage is attained, subsequent developmental characteristics are unrelated to the time of first cleavage.
B. Grimard, P. Humblot, A. A. Ponter, J. P. Mialot, D. Sauvant and M. Thibier
Effects of postpartum energy restriction, parity and time after parturition on energy status (measured by glucose, insulin, non-esterified fatty acids (NEFAs) and β-hydroxybutyrate), LH secretion and follicular growth were investigated in ten primiparous and nine multiparous suckled cows. Females were allocated by parity, body mass and body condition score at calving to diets supplying either 100% (CE, n = 10) or 70% (LE, n = 9) of energy requirements until day 70 postpartum. Metabolic parameters were measured every week from calving to day 70 postpartum. Blood samples were collected at intervals of 15 min for 10 h on day 30 and day 50 after parturition for LH measurement. Ovaries were examined between days 20 and 30 and days 40 and 50 postpartum by ultrasonography. Energy supply affected mean plasma concentrations of glucose (CE: 0.64 ± 0.01 g l−1 versus LE: 0.61 ± 0.01 g l−1; P < 0.05) and NEFA (CE: 168 ± 17 μeq l−1 versus LE: 309 ± 18 μeq l−1; P < 0.01) but by day 70 postpartum, glucose and NEFA concentrations were not significantly different between the two groups. LH pulse amplitude and frequency were not affected by energy supply (P > 0.10). However, at day 30 postpartum, LH pulse frequency was negatively correlated with plasma concentration of NEFA (r= −0.61; P < 0.01). Cows fed diets supplying 100% of energy requirements had more large follicles than did cows fed low energy diets (CE: 0.82 ± 0.05 versus LE: 0.31 ± 0.05; P < 0.05). The size of the largest follicle was greater in CE cows than in LE cows (CE: 10.2 ±0.1 mm versus LE: 8.7 ± 0.2 mm; P < 0.05). Between 40 and 50 days postpartum, the size of the largest follicle was negatively correlated with NEFA concentration (r= −0.5; P < 0.05). These results suggest that LH pulse frequency might be affected by energy supply when energy balance was strongly negative, whereas follicular growth was affected at a later stage, after parturition.
J. M. Wallace, R. P. Aitken, M. A. Cheyne and P. Humblot
The objective of this study was to investigate whether peripheral plasma profiles of pregnancy specific protein B (PSPB) are predictive of pregnancy outcome in adolescent sheep in which growth of the placenta has been compromised by the competing nutrient demands of maternal tissue synthesis. Embryos recovered on day 4 after oestrus from adult ewes inseminated by a single sire were transferred in singleton to prepubertal adolescent recipients. After transfer, the adolescent recipients were individually offered a high or low proportion of a complete diet to promote rapid (RMG) or normal (NMG) maternal growth rates (n = 12 per group). After day 100 of gestation the feed intake of the NMG group was adjusted weekly to meet the nutrient requirements of the gravid uterus. Blood was sampled three times a week throughout gestation and analysed for PSPB and progesterone. Liveweight gain during the first 120 days of gestation was 229 ± 9.1 and 105 ± 3.9 g day−1 for the RMG and NMG groups, respectively. For ewes delivering live young, mean placental mass at term was 263 ± 16.8 and 438 ± 44.6 g (P < 0.002), while lamb birthweight was 2.74 ± 0.25 and 4.34 ± 0.27 kg (P < 0.001) for the RMG (n = 8) and NMG (n = 11) groups, respectively. The biphasic pattern of PSPB secretion during gestation was similar in all ewes delivering live young, but individual concentrations within treatment groups were highly variable. Mean PSPB concentrations were lower in RMG than in NMG ewes throughout gestation (P < 0.05) and the major differences in relative terms were detected between days 50 and 100 of pregnancy. PSPB concentrations during this latter period were correlated (P < 0.05) with placental mass at term but not with lamb birthweight. High dietary intakes, leading to rapid maternal growth rates were associated with low peripheral progesterone concentrations (P < 0.02) throughout gestation. Irrespective of treatment group, progesterone concentrations during the second half of pregnancy were positively associated with both placental mass at term (P < 0.002) and lamb birthweight (P < 0.01). The incidence of non-infectious abortion during late gestation (125 ± 1.3 days) was higher (P < 0.001) in the RMG (4 of 12) than in the NMG (1 of 12) group and was associated with abnormal PSPB profiles in the former group. The mass of the fetus at the time of abortion was highly correlated (P < 0.01) with mean PSPB concentrations up to day 120 of gestation, but was independent of peripheral progesterone concentrations. These results suggest that sequential measurement of PSPB may provide a reliable indicator of fetal distress and adverse pregnancy outcome in singleton bearing ewes. PSPB and progesterone analysis may also have prognostic value as a biochemical marker of suboptimal placental growth and function in sheep.
P. Humblot, G. De Montigny, N. Jeanguyot, F. Tetedoie, B. Payen, M. Thibier and R. G. Sasser
Summary. The 34 French Alpine dairy goats originated from a single flock and were artificially inseminated 44 h after synchronization of oestrus. They were bled daily at the jugular vein from 15 to 27 days after AI. An early pregnancy diagnosis by RIA of progesterone concentration was performed 21 days after AI. In pregnant goats (≥ 1·5 ng progesterone/ml) daily sampling was extended until 30 days after AI and, from those, 9 were bled every 2 weeks until the end of pregnancy and at 50 and 63 days post partum. Pregnancy-specific protein B (PSPB) was also assayed.
The kidding rate was 67·6% (23/34). PSPB concentrations (ng/ml) in pregnant goats were significantly different from those of non-pregnant goats at 24 days after AI (0·82 ± 0·18 vs 1·78 ± 0·19; mean ± s.e.m.) and rose to 40 ng/ml at the end of pregnancy. From Day 25 and throughout gestation, females with 2 fetuses had higher PSPB concentrations than did those with a single fetus (P < 0·05). In the 2 goats exhibiting late embryonic mortality according to progesterone concentrations, one had a PSPB profile very similar to those of pregnant goats until 30 days while the other did not show any elevation of PSPB concentration. It is concluded that PSPB profiles in goats are similar to those found in cows throughout pregnancy and that PSPB RIA may be useful for pregnancy diagnosis or diagnosis of late embryonic mortality.
Keywords: PSPB; progesterone; pregnancy; embryonic mortality; goat
P. Humblot, S. Camous, J. Martal, J. Charlery, N. Jeanguyot, M. Thibier and R. G. Sasser
Summary. Pregnancy-specific protein B (PSPB) and progesterone concentrations were determined by RIAs in venous plasma during early pregnancy after 177 artificial inseminations (AI) performed in 76 cows and 71 heifers. The females were bled at 24, 26, 30–35 days and ∼ 70 days (for non-returns to oestrus) after AI. In non-pregnant females without extended CL maintenance (progesterone < 1·5 ng/ml on Day 24) and or showing a normal time of return to oestrus (Group 1, N = 63), PSPB concentrations were undetectable whatever the stage after AI except in 2 cows. In pregnant animals (N = 83; Group 2) progesterone concentrations were > 10 ng/ml from Day 24 to the time of rectal palpation and PSPB concentrations rose continuously from 0·42 ± 0·07 (s.e.m.) ng/ml (Day 24) to 4·06 ± 0·3 ng/ml (time of rectal palpation). No coefficient of correlation between PSPB and progesterone concentrations was significant whatever the day of gestation studied. In cows with extended luteal function and subsequently found to be non-pregnant (late embryonic mortality) PSPB was undetectable (N = 21; Group 3) or detectable (N = 10; Group 4) at Days 24, 26 and/or 30–35 of pregnancy. At 24 and 26 days after AI progesterone concentrations were intermediate between those of Groups 1 and 2. At Day 24 females of Group 4 had higher progesterone concentrations than those of Group 3 (P < 0·05), but no differences between these two groups existed at subsequent stages after AI. Animals of Group 4 had lower PSBP concentrations than those of Group 2 between Days 24 and 30–35 (P < 0·025) but at the time of rectal palpation PSPB values fell to undetectable levels in all but 1 cow of Group 4. We conclude that (1) most pregnancy failures in cows are due to nonfertilization or early embryonic death and if AI is performed after 70 days post partum >95% of these females have no detectable PSPB concentrations; (2) peripheral progesterone concentrations are lower at Days 24–26 after AI in cows with late embryonic mortality than in pregnant cows; (3) only 30% of non-pregnant females with extended luteal function (late embryonic mortality) have detectable PSPB levels which are lower than in pregnant cows; and (4) in pregnant animals there is no correlation between PSPB and progesterone concentrations. This suggests that under physiological conditions PSPB has no major effect on progesterone production or vice versa.
Keywords: PSPB; progesterone; pregnancy; embryonic death; cow
S Freret, B Grimard, A A Ponter, C Joly, C Ponsart and P Humblot
The aim of our study was to test whether a reduction in dietary intake could improve in vitro embryo production in superovulated overfed dairy heifers. Cumulus–oocyte complexes of 16 Prim’ Holstein heifers (14 ± 1 months old) were collected by ovum pick-up (OPU), every 2 weeks following superovulation treatment with 250 μg FSH, before being matured and fertilized in vitro. Embryos were cultured in Synthetic Oviduct Fluid medium for 7 days. Heifers were fed with hay, soybean meal, barley, minerals and vitamins. From OPU 1 to 4 (period 1), all heifers received individually for 8 weeks a diet formulated for a 1000 g/day live-weight gain. From OPU 5 to 8 (period 2), the heifers were allocated to one of two diets (1000 or 600 g/day) for 8 weeks. Heifers’ growth rates were monitored and plasma concentrations of metabolites, metabolic and reproductive hormones were measured each week. Mean live-weight gain observed during period 1 was 950 ± 80 g/day (n = 16). In period 2 it was 730 ± 70 (n = 8) and 1300 ± 70 g/day (n = 8) for restricted and overfed groups respectively. When comparing period 1 and period 2 within groups, significant differences were found. In the restricted group, a higher blastocyst rate, greater proportions of grade 1–3 and grade 1 embryos, associated with higher estradiol at OPU and lower glucose and β-hydroxybutyrate, were observed in period 2 compared with period 1. Moreover, after 6 weeks of dietary restriction (OPU 7), numbers of day 7 total embryos, blastocysts and grade 1–3 embryos had significantly increased. On the contrary, in the overfed group, we observed more <8 mm follicles 2 days before superovulation treatment, higher insulin and IGF-I and lower nonesterified fatty acids in period 2 compared with period 1 (no significant difference between periods for embryo production). After 6 weeks of 1300 g/day live-weight gain (OPU 7), embryo production began to decrease. Whatever the group, oocyte collection did not differ between period 1 and 2. These data suggest that following a period of overfeeding, a short-term dietary intake restriction (6 weeks in our study) may improve blastocyst production and embryo quality when they are low. However, nutritional recommendations aiming to optimize both follicular growth and embryonic development may be different.