J.S.M. Hutchinson

In vivo oocyte recovery and in vitro embryo production from bovine donors aspirated at different frequencies or following FSH treatment.

The effects of frequency of follicular aspiration and treatment of donor cattle with FSH on in vivo oocyte recovery and in vitro embryo production were studied. Simmental heifers (n = 24) formed 8 replicates of 3 treatments in which oocyte donors were aspirated 1) once a week, 2) twice a week, or 3) once a week following treatment with FSH for 3 d prior to aspiration. Oocytes were graded, washed, matured for 20 to 24 h and then inseminated with frozen/thawed semen from a single sire, followed by co-culture on granulosa cell layers. Embryo development was observed until Day 7 after insemination. Significantly fewer follicles per heifer per week were counted (14.7+/-2.3 vs. 27.4+/-3.1 vs. 23.1+/-2.8) and aspirated (12.0+/-2.0 vs. 21.8+/-2.7 vs. 20.1+/-2.6) in heifers on the once-weekly than twice-weekly aspiration treatment (P<0.01) or on the once-weekly aspiration after FSH treatment (P<0.05). There were no significant differences between treatments in the total number of oocytes recovered per week (5.6+/-1.2 vs. 8.9+/-1.5 vs. 6.1+/-1.2), but significantly more oocytes per heifer per week recovered from animals treated with FSH were graded Category 1 (2.8+/-0.4), i.e., >4 layers good cumulus with a clear, even cytoplasm, than from animals aspirated once (0.9+/-0.2; P<0.01) or twice a week (1.5+/-0.3; P<0.05). The number of transferable morulae plus blastocysts produced per heifer per week was higher from animals aspirated twice a week (2.4+/-0.4; P<0.05) or once a week following FSH treatment (2.1+/-0.4; P<0.05) than from animals aspirated once a week without FSH treatment (1.0+/-0.3). In conclusion, FSH treatment of bovine oocyte donors aspirated once a week enabled a similar number of transferable embryos to be produced per donor week as aspiration twice a week without FSH treatment. These 2 treatments produced twice as many transferable embryos per donor week as aspiration once a week without FSH treatment.

In vivo oocyte recovery and in vitro embryo production from bovine oocyte donors treated with progestagen, oestradiol and FSH

The effect of treatment of donor cattle with progestagen and oestradiol or FSH on in vivo oocyte recovery and in vitro embryo production was studied. Forty-eight beefxFriesian cows formed eight replicates of six treatments in a 2 (no steroid versus steroid)x3 (none, single or multiple dose(s) of FSH) factorial design in which follicles were aspirated once weekly for 3 weeks. Oocytes were graded, washed, matured for 20-24h and then inseminated with frozen/thawed semen from a single sire followed by coculture on granulosa cell monolayers. Treatment with steroid had no significant effect on any follicular, oocyte or embryo production variate other than to reduce the number (P<0.05) and the diameter of large follicles>10mm (P<0.01) present at aspiration. FSH increased numbers of medium (6-10mm) and large follicles (P<0.01) and there was a corresponding decrease in the number of small follicles (2-5mm; P<0. 01). The total number of follicles at aspiration increased from 17. 7+/-1.60 for animals not treated with FSH to 23.6+/-1.97 following multiple dose treatment with FSH (P<0.05). Significantly, more follicles were aspirated following FSH treatment (no FSH 9.7+/-1.09, single dose FSH 13.6+/-1.30, multiple dose FSH 17.3+/-1.52; P<0.05) and numbers of oocytes recovered per cow per week increased (no FSH 4.1+/-0.76, single dose FSH 5.3+/-0.87, multiple dose FSH 5.9+/-0. 94) but the differences were not significant. Significantly, more good oocytes (Category 1) were recovered from animals treated with FSH (P<0.05). There was no overall significant effect of FSH on embryo production rate or the total number of transferable embryos produced but the number of transferable embryos was highest following administration of multiple doses of FSH. In conclusion, progestagen plus oestradiol 17beta treatment did not affect follicle, oocyte and embryo production of oocyte donors aspirated once per week. FSH treatment, however, significantly increased the number of follicles aspirated and Category 1 oocytes recovered. Multiple dose administration of FSH resulted in the production of the highest number of transferable embryos but this effect was not significant.

Naloxone evokes a nutritionally dependent LH response in post partum beef cows but not in mid-luteal phase maiden heifers

Data from two experiments are reported which test the hypothesis that nutrient and/or dry-matter intake and body condition may interact to modify hypothalamic opioidergic activity and thus influence the pulsatile release of LH during the early post-partum period and during the oestrous cycle. Experiment 1 involved 16 multiparous, twin-suckling beef cows, and was a 2 × 2 × 2 factorial design in which the factors were level of post-partum energy intake (80 v. 130 M) metabolizable energy (ME) per day), the digestible undegradable protein (DUP) content of the post-partum diet (14 v. 31 g/kg dry matter), and treatment with either 200 mg or 400 mg naloxone hydrochloride. Blood samples were collected at 15-min intervals for 4h at weeks 4 and 7 post partum. Naloxone was administered intravenously after the eighth sample. Experiment 2 involved 16 cyclic maiden heifers and was also arranged in a factorial manner, with two levels of body condition at the start of the experimental period (2·50 and 3·16 units) and two levels of energy intake thereafter (40 and 80 MJ ME per day). Seven blood samples were collected at 15-min intervals on 4 days consecutively during the mid-luteal phase of the oestrous cycle. On the first 2 of these 4 days naloxone was administered, whilst on the last 2 days a gonadotropin-releasing hormone agonist (buserelin; GnRH) was administered, both after the fourth sample. Plasma from both experiments was assayed for LH and prolactin (Prl). In experiment 1, cows on 130 MJ ME per day returned to oestrus and ovulated earlier than cows on 80 MJ ME per day (44·5 v. 55·0 days; s.e.d. = 3·93; P v. 1·12; P v. 1·68; P Suckled cows given a high energy diet during the early post-partum period can overcome the opioid mediated block on LH release and resume oestrous cycles earlier than cows given a low energy diet. LH would appear to be inhibited by a non-opioid mechanism in mid-luteal phase heifers. Total pituitary reserves ofLH may be influenced by the animals nutritional status.

Follicular dynamics and superovulatory response in heifers

The study examined whether the response of heifers to exogenous gonadotrophin superovulatory treatment could be predicted from a knowledge of previous antral follicular dynamics. During a pretreatment monitoring phase, of 24 normal oestrous cycles (20.1 ± 0.33 days long) observed in 17 heifers, one, 15 and seven cycles showed one, two and three antral follicular waves respectively, as measured by ultrasonography. The subsequent ovulatory response (number of corpora lutea) to ovine FSH stimulation, after a CIDR-B/oestradiol benzoate/prostaglandin analogue cycle synchronisation regime, was not correlated with either oestrous cycle length or follicle wave number during the monitoring phase or with the number of follicles observed at the start of FSH treatment, but was related to the number of follicles observed during the monitoring phase (r = 0.47, P < 0.05). In conclusion, the present results show that the outcome of FSH superovulatory stimulation in heifers cannot be predicted from a knowledge of prior follicular dynamics.

Mechanisms linking under-nutrition and ovarian function in beef heifers

Prolonged reduction in energy intake in beef heifers has been reported to suppress ovulation but the mechanisms involved are poorly understood. The objective of this study was to examine whether changes in the pattern of LH secretion following each of three different tests predicted the functional state of the hypothalamo-pituitary-ovarian (H-P-O) axis. Test 1 examined the ratio of LH secretion during the 1h before and 2h after naloxone (NAL) administration. The other two tests assessed the LH surge following an exogenous oestradiol positive feedback signal (Test 2) or exogenous progesterone priming (Test 3). In phases 1 and 3, each of 8 weeks duration, the heifers were fed 100% of their maintenance energy requirements. In phase 2, of 9 weeks duration, they were fed 50% of their maintenance energy requirements. Oestrus was induced in all heifers by PG administration at the start of the experiment. Heifers were administered a naloxone challenge of 50, 100, 200 or 400mg naloxone hydrochloride i.v. (one dose per heifer) during the mid-luteal period of phase 1 and all four naloxone treated heifers received 400mg naloxone hydrochloride at the end of phases 2 and 3. Doses of 10, 20 or 40 mg oestradiol benzoate (EB) i.m. were each administered to two of the remaining heifers during the mid-luteal period of phase 1. One heifer on each dose of oestradiol benzoate in phase 1 had the same dose administered at the end of phases 2 and 3. The progesterone challenge was administered to three heifers by insertion of a PRID for 12 days starting in the middle of phase 2. In Test 1, the ratio of LH secretion before and after naloxone administration in phase 1 was 1:1 (50mg), 1:4 (100mg), 1:4 (200mg) and 1:9 (400mg) (50mg versus 100mg and 100mg versus 200mg doses, P<0.05); 50mg versus 400mg doses, P<0.001). In phase 2, this ratio was 1:1 and there was no response to 400mg dose of naloxone in any of the four heifers. In phase 3, the ratio depended on the ovarian activity in the heifer and ranged from 1:1 to 1:4 (P<0.05). In Test 3, a positive oestradiol feedback signal was detected in cyclic heifers in phases 1-3 but not in the acyclic heifer in phase 2. Heifers challenge with exogenous progesterone did not have oestradiol or LH values above threshold levels. We conclude that all three tests successfully predicted the functional state of the hypothalamo-pituitary-ovarian axis. In nutritionally undernourished beef heifers onset of ovarian acyclicity is either preceded or accompanied by the loss of a positive feedback signal (Test 2) and progesterone priming ability (Test 3), and that a plasma LH ratio of > or =1:2 following naloxone challenge (Test 1) is a sign of recovery of the functional state of the hypothalamo-pituitary-ovarian axis.

Follicle-stimulating hormone-stimulated differentiation and progesterone production of bovine granulosa cells in culture.

Abstract Progesterone production of granulosa cells cultured in vitro is stimulated and cell differentiation increased, by follicle-stimulating hormone (FSH). This study examined whether the increased progesterone production observed when bovine granulosa cells are cultured occurs because (1) progesterone production by undifferentiated and/or differentiated cells is increased or (2) the differentiation of granulosa cells is stimulated. Viable bovine granulosa cells (2−3×105) from follicles 5–8 mm in diameter were cultured in the presence of 0, 1, 10 and 100 μu FSH (1 μu ≡ 1 μg NIH-FSH-S1) for 6 days at 37°C in a humidified atmosphere of 5% CO2 in air in 1 ml of a 1:1 mixture of Dulbeccos modified Eagle medium: Hams F10 medium supplemented with 365 μg ml−1 l -glutamine, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin. Progesterone production, total DNA and protein, and cell diameter were determined sequentially over the culture period. The increases in progesterone production (ng μg−1 DNA per 24 h), cytoplasmic:nuclear ratio (μg protein μg−1 DNA) and cell diameter (μm) over 6 days culture indicated that granulosa cells underwent differentiation in the presence of FSH. Progesterone production of undifferentiated granulosa cells (diameter 14 μm or less) was stimulated by FSH (P 0.05) during a 6 day culture period. FSH stimulated (P 0.05) progesterone production by differentiating granulosa cells (8.7±0.5 ng μg−1 DNA per 24 h). In conclusion, the increase in progesterone production of FSH-stimulated granulosa cells cultured in vitro appears to be mainly due to an increase in the number of differentiating cells with a constant rather than an increasing progesterone production per cell.