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S. G. Revell and P. D. P. Wood

Summary. Bull spermatozoa were diluted in skim milk–egg yolk and frozen. After thawing, the samples were added to citrate buffer and photographed (1 sec exposure, 400 ASA, dark field) to identify the tracks of the moving spermatozoa.

The proportions of motile spermatozoa in 1707 photographs of semen samples from 25 ejaculates were distributed binomially, and allowed motility to be estimated at a predictable level of precision, and without bias when one photograph from each of two straws was taken at random from an ejaculate. The variance was equal to its expectation and inversely proportional to the total number of spermatozoa in each photographic field.

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A. P. Carter, P. D. P. Wood and Penelope A. Wright

Summary. The relationships between scrotal circumference and live weight and sperm output were examined in 3 samples of bulls selected for use in AI. In Study 1 the linear correlation between live weight and scrotal circumference in 418 British Friesian bulls at about 400 days of age was +0·28, and the circumference of the scrotum was related to the live weight of the bull by +0·179 ± 0·060 mm/kg live weight. At 400 days of age the mean (± s.e.) live weight and scrotal circumference of 22 British Friesian bulls in Study 2 was 412 ± 7 kg and 332 ± 4 mm respectively. Rates of growth were 1·11 ± 0·023 kg/day and 0·426 ± 0·023 mm/day. The correlation (+0.42) between the size of the scrotum and the no. of spermatozoa/ ejaculate was not significant. British Friesian (average age 9 years) and Hereford (average age 6·5 years) bulls were examined in Study 3. For the 25 Herefords, the correlation between the number of usable straws over a 6-month period and the scrotal circumference was +0.43, compared with +0.21 among the 28 British Friesians. It is concluded that scrotal circumference is unlikely to be an accurate predictor of sperm output in AI bulls.

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F. B. P. Wooding and D. Claire Wathes

Summary. The ultrastructure of cow placentomes, collected between 37 and 260 days of gestation, was examined. The microvillar junction and binucleate cell granules were selectively stained by phosphotungstic acid. Fetal binucleate cells interrupted the microvillar junction and penetrated as far as the basement membrane of the uterine epithelium. The uterine epithelium included not only binucleate cells which contained the distinctive granules but also non-granulated binucleate cells with pyknotic nuclei at the microvillar junction. Binucleate cells with pyknotic nuclei were also seen within chorion cells. It is suggested that the normal function of a mature chorionic binucleate cell at all the stages of bovine pregnancy is migration into the uterine epithelium to release its granules and subsequent condensation to a cell remnant which is phagocytosed by the chorionic epithelium.

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P. D. P. Wood, J. A. Foulkes, R. C. Shaw and D. R. Melrose

Summary. Nineteen young Hereford bulls were used to study the relationship between semen characteristics and fertility in artificial insemination following 15 320 inseminations. Seven measures of sperm motility, morphological abnormalities, the release of hyaluronidase, ATP content and sperm head measurements were examined as predictors of fertility (49-day fixed-interval non-return rate). Two assessments of motility, three categories of abnormal spermatozoa, acrosomal changes and the release of hyaluronidase had predictive power. Multiple regression analysis showed that a combination of sperm motility after dilution in saline, motility after thawing and the proportion of coiled tails and proximal protoplasmic droplets provided the best prediction of fertility and allowed bulls to be ranked in order of observed non-return rate (%) with a Spearman correlation better than +0·80.

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T. C. Wood, A. P. Byers, B. E. Jennette and D. E. Wildt

Immature eggs were recovered from freshly excised ovaries from domestic cats, and initially 931 eggs with a compact cumulus and uniform cytoplasm were cultured in 1 of 15 treatments. Eagle's minimum essential medium containing glutamine and pyruvate was supplemented with 5% (v/v) fetal calf serum (FCS), 4 mg bovine serum albumin (BSA) ml−1 or 2 mg polyvinyl alcohol ml−1 (PVA; non-protein control). Within each of these supplement groups, eggs were cultured with: no hormone; LH + FSH; LH + FSH + oestradiol; or LH + FSH + oestradiol + progesterone. After incubation for 52 h, eggs were inseminated with conspecific fresh spermatozoa, cultured and examined for stage of meiotic maturation and in vitro fertilization (IVF). There were fewer (P <0.05) eggs maturing to metaphase II in vitro in FCS compared with BSA or PVA, the last two treatments producing similar (P < 0.05) results. Gonadotrophins in concert with oestradiol or oestradiol + progesterone improved the incidence of maturation (P ≤ 0.01) compared with no added hormones. The incidence of fertilization and cleavage in vitro ranged from 5.2 to 33.9% and varied (P < 0.05) with hormone subtreatment. Adding FSH + LH + oestradiol consistently increased the incidence of IVF approximately twofold compared with controls with no added hormones. Although it inhibited the ability of eggs to reach metaphase II, FCS in the presence of gonadotrophins and oestradiol allowed > 60% of mature eggs to fertilize in vitro (P < 0.05, compared with PVA and BSA). Inhibitory effects on egg maturation were further evaluated by testing four FCS batches from three commercial sources against BSA. All FCS batches were clearly inhibitory, as 60.7% of treated eggs arrested at the germinal vesicle stage (21.1% of BSA-treated eggs, P < 0.001). Dialysis of FCS eliminated a significant proportion of the inhibition; 69 of 158 eggs (43.7%) matured compared with 22 of 135 (16.3%) in complete or 23 of 160 (14.4%) in recombined serum. In summary, FCS appears to exert a paradoxical effect in this system, inhibiting maturation in vitro (apparently due to small molecular weight components), but promoting IVF of mature eggs if gonadotrophins and oestradiol are present. Although hormone supplementation enhanced the ability of in vitro matured eggs to fertilize and cleave, neither protein source nor the hormone treatments tested here appear responsible for the consistently low incidence of fertilization in cat eggs matured in vitro.

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S McMullen, J C Osgerby, L M Thurston, T S Gadd, P J Wood, D C Wathes and A E Michael

In the placenta, cortisol is inactivated by NADP+- and NAD+-dependent isoforms of 11β-hydroxysteroid dehydrogenase (11βHSD). Decreased placental 11βHSD activities have been implicated in intrauterine growth restriction (IUGR) and fetal programming of adult diseases. The objective of this study was to investigate whether placental 11βHSD activities and fetal plasma cortisol:cortisone ratios could be affected by nutritional restriction of ewes (70% maintenance diet) throughout gestation, for specific stages of gestation, or prior to mating. Chronic nutritional restriction from day 26 of gestation onwards decreased NAD+-dependent 11βHSD activities by 52 ± 4% and 45 ± 6% on days 90 and 135 of gestation respectively. Although the decreases in enzyme activities were associated with fetal IUGR, the cortisol:cortisone ratio in fetal plasma was unaffected by chronic nutritional restriction throughout pregnancy. Nutritional restriction confined to early (days 26–45), mid- (days 46–90) and late gestation (days 91–135), or the 30 days prior to mating, had no significant effect on NAD+-dependent, placental 11βHSD activities, nor was there evidence of IUGR. However, nutritional restriction at each stage of pregnancy and prior to mating was associated with significant decreases in the fetal plasma cortisol:cortisone ratio (3.2 ± 0.7 in control fetuses; 1.0 to 1.6 in fetuses carried by nutritionally restricted ewes). We conclude that nutritional restriction of pregnant ewes for more than 45 consecutive days can significantly decrease NAD+-dependent placental 11βHSD activities in association with IUGR. While the cortisol:cortisone ratio in fetal plasma is sensitive to relatively acute restriction of nutrient intake, even prior to mating, this ratio does not reflect direct ex vivo measurements of placental 11βHSD activities.

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S Wilsher, F Stansfield, R E S Greenwood, P D Trethowan, R A Anderson, F B W Wooding and W R Allen

Gross, histological and immunocytochemical examinations carried out on maternal and fetal reproductive tissues from two pregnant giraffes at an estimated 8 and 13.5 months of gestation (term=15 months) revealed a typically ruminant macrocotyledonary placenta with binucleate trophoblast cells scattered sparsely in the placentome where they stained intensely with a prolactin antiserum. Binucleate cells were present in greater numbers in the intercotyledonary allantochorion where they did not stain for prolactin whereas the uninucleate trophoblast still did. A single large corpus luteum of pregnancy and several small luteinised follicles were present in the maternal ovaries while the fetal ovaries at 13.5 months gestation showed an assortment of enlarging antral follicles and partially and completely lutenised follicles, the granulosa and luteal cells of which stained positively for 3β-hydroxysteroid dehydrogenase (3β-HSD), 17,20 lyase, prolactin, progesterone receptor and androgen receptor, but negatively for aromatase. The uninucleate trophoblast of the placentome and intercotyledonary allantochorion, the epithelium of the maternal endometrial glands, the seminiferous epithelium in the fetal testis at 8 months of gestation and the zonae fasciculata and reticularis of the fetal adrenal at 13.5 months also stained positively for 3β-HSD and negatively for aromatase. Endocrinologically, it appears that the giraffe placenta is more similar to that of the sheep than the cow with a placental lactogen as the likely driver of the considerable degree of luteinisation seen in both the maternal and the fetal ovaries.