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R. G. WALES and J. C. WALLACE

In a recent communication (Wales & Wallace, 1964), it was observed that the oxidation of substrates other than fructose was greatly depressed by washing the spermatozoa free of their seminal plasma. This effect could be due either to the removal of protective macromolecules in the seminal plasma or to the removal of alternative substrates. The following experiments were undertaken to investigate these possibilities. Veronal-buffered saline (pH 7·2) was the basic diluent used and the methods for preparing washed suspensions, for incubating the spermatozoa, for the assay of radioactivity and for the estimation of lactate and fructose, have been previously described (Wallace & Wales, 1964; Wales & Wallace, 1964). Sorbitol was estimated by an enzymic method (T. O'Shea and R. G. Wales, unpublished data). Acetate was estimated after steam distillation of metaphosphoric acid extracts (Annison, 1954). The
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R. G. WALES and J. C. WALLACE

Summary.

A study has been made of the effects of potassium, magnesium, calcium and phosphate ions and their possible inter-relationships on the metabolism of bull, dog, rabbit and fowl spermatozoa. Respiration and fructolysis were measured and isotopically labelled fructose used to assess the contribution of fructose oxidation to total oxygen uptake.

Overall, fowl spermatozoa had the lowest rate of metabolism. Dog spermatozoa had the greatest respiratory activity and differences in oxygen uptake between the mammalian species were due mainly to variations in the rate of fructose oxidation. Washing bull, dog and rabbit spermatozoa greatly reduced the oxidation of substrates other than fructose, but washing had little effect in the fowl.

In general, phosphate ions depressed the respiration of bull spermatozoa, but stimulated their aerobic fructolysis. Except for stimulating the respiration of unwashed dog spermatozoa, phosphate had no important influence on metabolism in the other species. Potassium stimulated some aspects of metabolism in all species, but had its greatest effect upon the respiration of dog spermatozoa. Magnesium depressed the respiration of bull and fowl spermatozoa, but calcium was without effect. There were few significant interactions between the ions tested.

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J. C. WALLACE and A. K. LASCELLES

Summary.

An analysis of jugular blood plasma and testicular lymph collected from conscious rams has been carried out. The white cell content of the testicular lymph was 100 to 400 cells/μl and most of the cells were medium lymphocytes. In general the chemical composition of the testicular lymph was similar to that of the lymph collected from other regions of the body of sheep and other animals. The unusual composition of testicular and epididymal fluids thus was not reflected in the composition of the lymph. There was a large plasma-lymph gradient for lactate and glucose suggesting that these substances are utilized by testicular tissue. The free fatty acid level in lymph was somewhat lower than in plasma.

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J. C. WALLACE and R. G. WALES

Summary.

The effects of potassium, magnesium, calcium and phosphate ions on the metabolism of ejaculated and epididymal ram spermatozoa have been studied. Respiration and fructolysis were measured and isotopically labelled fructose was used to assess the contribution of fructose oxidation to total oxygen uptake.

Potassium and phosphate ions significantly increased both respiration and fructolysis; magnesium and calcium had much less influence and there were few significant interactions between the ions tested.

The most significant effect of washing spermatozoa free of seminal plasma was the reduction in the oxidation of substrates other than fructose. Washing also tended to accentuate the effects of other treatments.

There were differences between ejaculated and epididymal spermatozoa in the response of oxidative metabolism to potassium but, in general, the metabolic patterns of these cells were similar.

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J. C. WALLACE and I. G. WHITE

Summary.

The occurrence of an enzyme which hydrolyses seminal glycerylphosphorylcholine (gpc) has been demonstrated in uterine rinsings of the ewe, cow, sow, rat and mouse. The products of the breakdown of gpc by rinsings of the ewe's uterus, which also contain an active phosphatase, are glycerol, inorganic phosphate and choline. Lower levels of gpc diesterase activity were also found in the cervical and oviduct fluids of the ewe. No diesterase activity could be demonstrated in the follicular fluid of the ewe, which contained appreciable concentrations of lactic acid and glucose.

Secretion of the diesterase in the ewe is influenced by the stage of the oestrous cycle independently of changes in other luminal fluid proteins or the wet weight of the uterus. Diesterase activity in uterine rinsings of the ewe is greatest at the time of ovulation.

The evidence indicates that the enzyme is a product of the uterine tissue and is not of bacterial origin. The optimal pH is 7·7 and the enzyme is not inhibited by fluoride or eserine.

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B. M. N. Wallace and J. B. Searle

Female common shrews in their first pregnancy were collected near Oxford (UK) from the hybrid zone between the Oxford and Hermitage races. These races differ by Robertsonian chromosomal rearrangements and both Robertsonian heterozygotes and homozygotes are found in the zone. The three homozygotes examined in this study had significantly more oocytes than the six heterozygotes. Among the heterozygotes, the number of oocytes tended to be lower in more severely heterozygous individuals; one double heterozygote was particularly depleted. Although there were, on average, 41% fewer primordial follicles in the ovaries of the heterozygotes than those of the homozygotes, there was no significant difference in the numbers of growing follicles between the karyotypic classes. These data suggest that Robertsonian heterozygotes may have a shorter reproductive lifespan than do homozygotes, but the numbers of follicles being recruited for ovulation at a particular instance during the fertile period does not appear to differ between homozygotes and heterozygotes. Morphological differences between the follicles of homozygotes and heterozygotes were not detected. Only 0.12% of healthy growing follicles were biovular.

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J. M. Wallace, J. J. Robinson and R. P. Aitken

Summary. 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 × 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 > 150 days. Pregnancies were successfully established in 9 recipient ewes, resulting in the birth of 10 viable lambs.

Prolactin concentrations were significantly higher (P < 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.

Keywords: post partum; fertilization; embryo viability; pregnancy; sheep

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J. J. Robinson, Jacqueline M. Wallace and R. P. Aitken

Summary. In Exp. 1, 40 ewes were used in a 2 × 2 factorial design to investigate the effects of intrauterine versus cervical insemination and superovulation using pig FSH or PMSG and GnRH on egg recovery and fertilization rate. Cervical inseminations were carried out at 48 and 60 h (N = 20 ewes) and intrauterine insemination at 52 h (N = 20 ewes) after progestagen pessary withdrawal. Eggs were recovered on Day 3 of the oestrous cycle. Ovulation, egg recovery and fertilization rates were independent of the type of superovulatory hormone used. Fertilization rate was high irrespective of insemination site but intrauterine insemination at 52 h was associated with a significant (P < 0·01) decrease in egg recovery of over 40% compared with cervically inseminated ewes.

In Exp. 2 ewes were inseminated at 36 (N = 5), 48 (N = 6) or 60 (N = 6) h after pessary withdrawal to determine the optimum intrauterine insemination time to maximize both fertilization rate and egg recovery. Egg recovery per ewe flushed was 23, 59 and 67% after intrauterine insemination at 36, 48 and 60 h respectively. Correspondingly, 0, 85 and 100% of the eggs recovered were fertilized. The results of Exps 1 and 2 suggest that when intrauterine insemination occurs before or during ovulation it interferes with oocyte collection by the fimbria.

In Exp. 3 egg recovery and fertilization rates were determined after cervical insemination at 48 and 60 h (N = 8) or intrauterine insemination at 48 (N = 9) or 60 (N = 8) h after progestagen withdrawal. Ewes in the last two groups were subdivided and inseminated unilaterally or bilaterally. Egg recovery was high after cervical insemination (95%) but only 36% of these eggs were fertilized. Unilateral intrauterine insemination was as effective as bilateral in ensuring high fertilization rates (100 versus 97%). Intrauterine insemination at 48 h compared with 60 h resulted in a significantly lower (P < 0·05) percentage of eggs recovered (42 versus 90% respectively). However, reducing the degree of interference by adopting unilateral rather than bilateral insemination did not alleviate the detrimental effects of the 48-h insemination time on egg recovery.

From these results we advocate the adoption of intrauterine insemination at 60 h after progestagen withdrawal to maximize fertilization rate and egg recovery in superovulated ewes.

Keywords: superovulation; intrauterine insemination; fertilization; ovum recovery; ewe

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Jacqueline M. Wallace, J. J. Robinson and R. P. Aitken

Summary. In Exp. 1 the effect of lactation versus early weaning on luteal function was examined in seasonally anoestrous Finn Dorset ewes that were induced to ovulate at 21 (N = 14) or 35 (N = 14) days post partum by using a CIDR device and PMSG. Prolactin concentrations were significantly higher (P < 0·001) in lactating compared with early weaned ewes throughout the study. The proportion of lactating ewes with inadequate luteal function (as assessed by daily progesterone concentrations) in the 21-day group was 0·43 (3 or 7) compared with 0·67 (4 of 6) for those weaned within 2 days after parturition. Corresponding values for the 35-day group were 0 (0 of 4) and 0·14 (1 of 7) respectively. There was no evidence of abnormal luteal function in standard ewes (N = 8) for which the interval from parturition was > 150 days.

In Exp. 2 we examined whether pregnancy can be successfully established during the breeding season following transfer of embryos into lactating or early weaned ewes in the early post-partum period. Embryos were donated from Border Leicester × Scottish Blackface ewes for which the interval from previous parturition was > 150 days. These embryos were transferred synchronously on Day 5 after behavioural oestrus to recipient ewes with the same breeding history as the donors (standard ewes, N = 15) or to lactating or early weaned recipients that had been induced to ovulate on Day 21 (N = 16) or 35 (N = 24) post partum. In the 21-day group inadequate luteal function was observed in 2 of 7 (0·28) lactating and 4 of 9 (0·44) early weaned ewes compared with corresponding values of 1 of 13 (0·08) and 2 of 11 (0·18) in the 35-day post-partum group. Luteal function was normal in all standard ewes. The proportion of successful pregnancies in the standard ewes was 0·80 (12 of 15) compared with 0 in lactating and early weaned ewes in the 21-day group and 0·08 (1 of 13) and 0·36 (4 of 11) respectively in the 35-day group.

The incidence of inadequate luteal function is therefore independent of the suckling stimulus and is higher in ewes induced to ovulate on Day 21 than Day 35 post partum during breeding and non-breeding seasons. For early post-partum recipient ewes with normal luteal function it is suggested that the high incidence of pregnancy failure after transfer of embryos may be due to embryo mortality caused by an inappropriate uterine environment or the inability of the embryo to sustain its luteotrophic signal.

Keywords: post partum; corpus luteum; embryo transfer; pregnancy; uterus; ewe

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R. G. WALES, J. C. WALLACE and I. G. WHITE

Summary.

Analyses were made of fluid collected from the rete testis and the caput, corpus and cauda epididymidis of the bull. Both the major electrolytes and some organic constituents in the fluids were examined. There was a decrease in sodium and chloride as the fluid passed from the testis through the epididymis and at the same time there was a general increase in organic constituents. In testicular fluid, sodium and chloride made up the bulk of the ions present. In the epididymis, however, potassium replaced approximately half of the sodium and at the same time there was a general decrease in total electrolytes. Of the organic constituents, orcinol-reactive carbohydrate, protein and acidsoluble phosphorus, especially glycerylphosphorylcholine, were present in much higher concentrations in the epididymal than in the testicular fluid. Although little reducing sugar was found, lactic acid was present in all the fluids examined.