One-cell or two-cell rabbit embryos were cultured in protein-free media with various NaCl concentrations and osmolarity to determine relative sensitivity of embryos to changes in media composition. Embryos from replicates of donor rabbits were distributed randomly across treatments and cultured at 39°C. Zygotes were cultured in Expts 1, 2A and B, and 3, and two-cell embryos were cultured in Expts 4A and 4B. In Expt 1, blastocyst formation and number of cells were highest (P < 0.05) in the control medium with 93 mmol NaCl l−1 (270 mosmols) compared with media containing 63 and 116 mmol NaCl l−1 (220 and 316 mosmols). In Expt 2, embryos were cultured in media with 70 or 93 mmol NaCl l−1, varying in osmolarity from 250 to 320 mosmols by adding sorbitol. In media with 70 mmol NaCl l−1 and osmolarities of 250, 280 and 300 mosmols, there were 41, 56 and 50% expanded blastocysts, respectively (P < 0.05). With 93 mmol NaCl l−1 and osmolarities of 270, 293 and 320 mosmols, embryos developed into 37, 53 and 27% expanded blastocysts, respectively, (P < 0.05). In Expts 3A and 3B and 4A and 4B, the osmolarity of the medium was maintained at 270 mosmols by adding sorbitol to media containing 40 or 60 mmol NaCl l−1, and other components were reduced in media containing 100 and 116 mmol NaCl l−1 to compensate for the higher NaCl. Zygote development into blastocysts was greatly suppressed (P < 0.05) in media with 40, 60, 100 and 116 mm NaCl l−1, compared with the control (93 mmol NaCl l−1), whereas development of two-cell embryos into blastocysts was much less affected. These results appear to reflect a direct sodium chloride as well as osmolarity effect on embryo development; and zygotes are much more sensitive to these effects than are two-cell embryos.
W. HOLTZ and R. H. FOOTE
Cannulation of the ductus deferens for collection of epididymal spermatozoa has been performed successfully in rams, bulls and boars (White, Larsen & Wales, 1959; Amann, Hokanson & Almquist, 1963; Bennett & Rowson, 1963; Tadmor, Schindler & Kempenich-Pinto, 1969; Wierzbowski & Wierzchos, 1969). Our attempts to adapt reported procedures directly to the rabbit failed because the cannula frequently became blocked soon after its insertion. A branched cannula was developed which consisted of a silicone rubber tube (Silastic, Dow Corning) 30 cm long, 1·02 mm i.d. and 1·65 mm o.d. A smaller tube (0·64 mm i.d. and 1·19 mm o.d.) of the same material was connected laterally 3 to 5 mm from the end of the cannula that was to be inserted into the ductus deferens (Text-fig. 1). This arrangement permitted flushing, and reduced the time spermatozoa
EVA BROWN and R. H. FOOTE
Recently there has been considerable interest in the use of intra-uterine contraceptive devices, but their mode of action is unknown. Much of the successful interference with pregnancy in laboratory animals has involved the placement of sutures into the uterine wall (Doyle & Margolis, 1963, 1964; Eckstein & Adams, 1964; Marston & Chang, 1964; Zipper, Garcia & Pastene, 1964; Adams & Eckstein, 1965a, b; Kar & Kamboj, 1965). In the present experiments the plastic devices were simply inserted into the lumen of the uterus, so as to allow some freedom of movement. This parallels their use in humans, and avoids possible complications in interpreting results where sutures penetrate the uterine wall. The experiments were designed to determine both implantation and kindling rates and to note the effect of the devices on the corpora lutea, since foreign
R. R. MAURER and R. H. FOOTE
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.
R. R. MAURER and R. H. FOOTE
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.
R. R. MAURER and R. H. FOOTE
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.
I.-H. Bae and R. H. Foote
Summary. Rabbit oocytes from follicles ≥ 1 mm in diameter were cultured for 18 h in a defined medium with osmolality adjusted in 20 mosmol increments from 230 to 350 mosmol by altering only the NaCl concentration. Adjustment, based upon determination of the osmolality of the medium, was necessary because a difference existed between calculated and achieved osmolality in this complex solution. The proportions of oocytes which matured to meiosis II with polar body formation were 64, 68, 64 and 65% in media of 250, 270, 290 and 310 mosmol, respectively.
S. K. PAǓFLER and R. H. FOOTE
Forty-three males were divided into three experimental groups and ligatures placed unilaterally as follows: (1) on the ductus deferens, (2) on the ductus deferens and corpus epididymidis, and (3) on the ductus deferens, corpus epididymidis and ductuli efferentes. Semen was collected six times a week from all males before ligation and for as long as 12 weeks thereafter.
Spermatozoa transported normally from the caput to the cauda epididymidis in non-ligated controls were characterized by rapid migration of the protoplasmic droplets, a decrease in swollen acrosomes and other abnormalities, an increase in the percentage of motile cells and a striking increase in fertility. Ligation of the ductus deferens only had little effect upon any of these changes. Also, considerable motility and fertility was maintained for 12 weeks following single ligation in contrast to a reduction after 4 weeks in the group with the isolated cauda epididymidis. This suggests that considerable mixing of spermatozoa normally can occur in the cauda. The proportion of abnormal forms, particularly decapitated spermatozoa, increased considerably in the isolated cauda by 8 weeks.
In the isolated caput abnormal spermatozoa increased rapidly and motility decreased. The protoplasmic droplet movement was delayed, as 54% had droplets on the midpiece after 4 weeks in contrast with 16% on spermatozoa which migrated normally to the cauda. Severe degeneration and disappearance of spermatozoa followed after 4 weeks, indicating that the caput may have dissolution properties.
Litter size, based on all does inseminated, averaged only 0·5 for caput spermatozoa compared with 5·0 for both caudal and ejaculated spermatozoa. The morphological and fertility data indicate that extrinsic factors as well as intrinsic ones are required for complete development of the fertilizing capacity of rabbit spermatozoa.
IN-HA BAE and R. H. FOOTE
Progesterone stimulated oocytes to develop more rapidly in culture. The time-dependent effect was more pronounced on large preovulatory Graafian follicles than on small- and medium-sized follicles. Treatment with LH had no effect.
GARY B. ANDERSON and R. H. FOOTE
Department of Animal Science, and Division of Biological Sciences, Cornell University, Ithaca, New York 14853, U.S.A. (Received 4th February 1975)
In 1947, Chang successfully stored young rabbit embryos for several days at 10°C. Recent interest in the practical application of embryo transfer techniques to livestock has resulted in renewed efforts to preserve mammalian embryos by refrigeration (Sreenan et al., 1970; Kardymowicz, 1972; Moore & Bilton, 1973; Anderson & Foote, 1975b), and by freezing (Wilmut & Rowson, 1973; Bank & Maurer, 1974; Whittingham & Whitten, 1974).
Anderson & Foote (1974, 1975a) reported that embryo metabolism is reduced while embryos are held at 10°C, but substrate utilization and normal DNA, RNA and protein synthesis is restored by rewarming to 37°C. The experiments reported in this paper were conducted to compare the relative development of embryos in vitro and in vivo following storage at low temperature.
Two-celled embryos from mature Dutch-Belted rabbits induced