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R. R. MAURER and R. H. FOOTE

Summary.

Embryos from ageing (20 to 148 weeks of age) and young (20 to 30 weeks of age) donors were transferred to ageing (52 to 221 weeks of age) and young (18 to 30 weeks of age) recipients to partition the effects of ageing oocytes and uterine environment on embryo mortality. More than 3300 two- to eight-cell embryos collected following superovulation at six 6-month intervals were transferred. The average number of ovulation points per ageing donor doe superovulated at the six intervals declined with age and repeated superovulation. The average number of ovulations for the young donors at the six intervals also differed slightly, the low number (forty-one) for the last group possibly being due to the crossbred strain used. Both embryo recovery and cleavage rates usually exceeded 80% and did not differ between young and ageing donors. The percentage of viable young developing from embryos transferred from ageing and young donors showed that the potential for embryo development had not been impaired during 3 years of ageing. The percentage of viable young developing from embryos transferred to ageing and young recipients indicated that conditions for maintaining pregnancy had been impaired in the ageing recipients. The average number of ovulations for a group of old does superovulated for the first time at 229 weeks of age was fourteen compared to sixty-two for young controls, and only 26% of the embryos transferred from the old does developed into neonates, whereas 45% of those from young donors developed normally. As the female ages, the uterine environment may become less conducive to prenatal development and the oocytes then show the effects of the ageing process directly or as a result of exposure of the oocyte or young embryo to the ageing oviduct. At laparotomy 12 days after transfer, the ageing recipients had 45·3% pre- and 14·8% postimplantation mortality. Corresponding values for young recipients were 33·5 and 16·0% respectively. Laparotomy increased embryo mortality in ageing females only. The percentage of embryos which developed to blastocysts in vitro paralleled the development in vivo of embryos in ageing and young donors. The overall sex ratio of 81·6 males/100 females resulting from transferred embryos was significantly different (P<0·05) from the expected figure.

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R. R. MAURER and R. H. FOOTE

Summary.

Collagenase activity was measured 66 to 75 hr after parturition in does at 34, 167 and 204 weeks of age. Uteri of ageing does contained less collagenase than those of young does. Uterine collagen content tended to increase with age. At Day 12 of pregnancy, uterine acid-soluble collagen was higher in does 174 weeks old than in does at 38 weeks of age.

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R. R. MAURER and R. H. FOOTE

Summary.

Progesterone and 20α-hydroxypregn-4-en-3-one (20α-P) synthesis in vivo and in vitro was measured 12 days post coitum in does averaging 32 weeks (young) and 214 weeks old (ageing). Young pregnant does had an average of 7·7 corpora lutea and 7·3 implantations for a mortality rate of 4·3%. Similar values for ageing does were 7·7, 5·4 and 29%, respectively. The progesterone content of ovarian venous blood was higher in young pregnant does than ageing does but the 20α-P content did not differ. Administration of lh did not significantly alter progesterone blood levels in either age group but increased 20α-P levels in both groups. Synthesis of progesterone and 20α-P by interstitial tissue in vitro did not differ significantly between ages. Addition of lh to the incubation medium stimulated synthesis of progesterone by tissue from the ageing group whereas 20α-P synthesis was increased in the young group. There was a positive relationship between doe age and pituitary weight. The ageing does had heavier pituitary glands than the young does but there was no difference in total lh content.

A lower concentration of lh was found in the pituitary gland and a lower progesterone content in the ovarian venous blood of old compared to young pregnant rabbits. While these differences may be partly responsible for the lowered reproductive efficiency of the ageing female, the evidence is somewhat equivocal since total pituitary lh and synthesis of progesterone in vitro were similar in the two age groups.

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R. R. Maurer and H. M. Beier

Summary. The effects of isolated protein fractions from rabbit uteri (prealbumin, albumin, uteroglobin, and β-glycoprotein), unfractionated uterine proteins, progesterone, oestradiol-17β, and prostaglandin F-2α on the development of rabbit embryos in vitro were investigated. When exposed to individual protein fractions obtained from Day-6 uteri, 8-cell embryos did not develop into early blastocysts; morulae readily developed into early blastocysts, but further development was retarded. Progesterone (10−5–10−11 M) and prostaglandin F-2α (0·1–10 ng/ml) added to the medium slowed development of blastocysts to advanced stages. Growth of 8- to 16-cell embryos, morulae, and Day-4 blastocysts was stimulated by unfractionated uterine proteins obtained from Day-5 uterine flushings.

Although embryos cultured in medium containing BSA had similar rates of blastocyst formation and, ultimately, similar blastocyst expansion as did the embryos cultured in medium with unfractionated proteins, the radial and immediate expansion of the early blastocysts cultured in the latter approximated that found in utero.

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HIDEO ONUMA, R. R. MAURER and R. H. FOOTE

Successful in-vitro culture of fertilized mammalian ova greatly facilitates the study of embryo development. Since Brachet (1912) reported culturing 5-day rabbit blastocysts, considerable effort has been devoted to the development of an in-vitro culture system for rabbit ova. Several investigators have reported culturing 1- and 2-celled rabbit ova to the morula stage, but not to the blastocyst stage (Lewis & Gregory, 1929; Smith, 1949; Chang, 1950; Purshottam & Pincus, 1961; Daniel, 1964; Edwards, 1964). The mouse ovum is obstinate in early cleavage stages, but Brinster (1963) recently has successfully cultured 2-celled ova to the hatched blastocyst stage. The peculiar difficulty for the rabbit ova may involve the presence of a relatively tough zona pellucida and mucin coat as pointed out by Pincus (1936).

The present study was initiated to explore the possibility of in-vitro development of rabbit ova from predominantly 2- and 4-cell stages to the

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N. B. VARIAN, R. R. MAURER and R. H. FOOTE

Summary.

The superovulatory response of Dutch-belted does receiving 0·10, 0·25, 0·50, 1·00, 2·00 and 4·00 mg of lh/kg of body weight following priming with twice daily injections of 0·50 mg of fsh each for 3 days was examined. The does were inseminated immediately following lh injection. Rabbits given the lowest level of lh did not ovulate, and an average of 1·0 ovulation points was obtained following the 0·25 mg level of lh. All does receiving ≥0·50 mg of lh ovulated, with an average of 33·7, 40·0, 45·1 and 35·6 ovulation points for the 0·50, 1·00, 2·00 and 4·00 mg levels, respectively (P>0·10). Some large unruptured follicles remained in all groups after they received lh.

The proportion of cleaved ova collected from fsh-primed does 26, 28 and 30 hr after lh administration was 68, 86 and 91% respectively, whereas in non-primed controls all fertilized ova appeared to have cleaved by 26 hr. This difference is attributed to the longer time required for ovulation to be completed in fsh-primed animals rather than a delay in cleavage rate following fertilization.

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R. R. MAURER, HIDEO ONUMA and R. H. FOOTE

Summary.

Rabbit embryos in the two- and four-cell stage were placed in rabbit and bovine serum with and without the addition of 100 mg glucose per 100 ml serum and cultured for 97 hr. Maximum development in culture was to the expanding blastocyst stage. Embryos were transferred to recipient does after 0, 24, 36, 48, 62, 72, 88 and 97 hr in culture. Embryos transferred after 62 hr in culture readily developed into neonates, and one embryo cultured for 88 hr to the blastocyst stage developed into a full-term young. The addition of glucose significantly increased (P<0.005) the number of blastocysts produced in culture.

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R. R. MAURER, W. L. HUNT and R. H. FOOTE

Summary.

Repeated superovulation and in-vivo collection of ova were carried out with thirty does divided into three groups of ten does each. One group received fsh—lh, a second group received pmsg—hcg and the controls received only lh. Injections of fsh—lh at three 16-week intervals followed by one 8-week interval resulted in 46·5, 35·4, 25·2 and 18·0 ovulation points/doe. This decrease with time was significant (P<0·05). Corresponding values for the pmsg—hcg group which, however, received fsh—lh for the final superovulation were 13·6, 5·7, 6·2 and 20·3. The lh controls averaged 7·8, 7·0, 5·1 and 5·6 ovulation points. Overall treatment differences were highly significant (P<0·005). From the 1920 ovulation points 1593 ova (83%) were recovered, of which 83·1% were cleaved. Young born from unrecovered ova accounted for 3·1%.

Control kindlings by the same does at regular periods resulted in normal litter size but in fewer does kindling as the experiment progressed. Results of two bio-assays for antihormones suggested that this decrease was due to hormonal refractoriness which was most pronounced in the pmsg—hcg group.

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R. R. Maurer, G. F. Stranzinger and S. K. Paufler

Summary. Rabbit spermatozoa stored at 37°C for 5 hr, 5°C for 5 hr or 5 days, and −196°C for 5 hr, 5 days, or 1 year were used to artificially inseminate does induced to ovulate normal or excess numbers of eggs. Fertility and subsequent embryonic development were examined. Frozen spermatozoa stored for 5 hr fertilized fewer oocytes than those stored for 5 days or longer. Fewer embryos were recovered on Day 6 than on Day 2. Blastocysts were smaller when spermatozoa were stored for 5 days, were frozen, or were placed in superovulating does, but no statistically significant differences in the % of fetal survival were found in the groups of normally ovulating does. Embryonic wastage associated with storage of spermatozoa resulted from a reduced fertilization rate and/or increased embryonic mortality before implantation.

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G. F. STRANZINGER, R. R. MAURER and S. K. PAUFLER

Summary.

Rabbit semen was extended in a tris-yolk-12·5% DMSO extender and frozen in liquid nitrogen vapour or on a 'dry ice' block after glycerol had been added. No differences (P>0·10) were found between inseminations with liquid semen and semen frozen in pellets with regard to the number of young born or the pregnancy rate. Other methods produced significantly (P<0·05) fewer young. Significantly (P<0·05) more young were born to does inseminated 5 hr after they received the ovulating hormone.