The crossreactivity between bull spermatozoa and monoclonal antibodies initially raised against mouse spermatozoa and human spermatozoa was tested by indirect immunofluorescent assay. The three anti-human spermatozoa monoclonal antibodies examined (HSK-9, HS-11, HS-63) crossreacted with methanol-fixed bull spermatozoa, whereas the anti-mouse spermatozoa monoclonal antibodies (MS-4 and MS-7) did not. A separate experiment was conducted to determine the binding ability of HSK-9, HS-11 and HS-63 with live (fresh) bull spermatozoa incubated (39°C in CO2 incubator) in a capacitation medium (modified Tyrode's supplemented with 10 μg heparin ml−1). The binding of the monoclonal antibodies to the intra-acrosomal antigens of live bull spermatozoa was determined at 0, 2, 4, 6 and 8 h of incubation. At the beginning of incubation, binding was minimal (3.2 ± 1.7%), but a much higher percentage of spermatozoa exhibited fluorescent staining after 2 h. The maximal binding was observed after incubation for 8 h (72.0 ± 8.2%). The third experiment was performed to determine binding of HS-11 to frozen–thawed spermatozoa and to test whether there was any variation among bulls in HS-11 binding to spermatozoa, and to assess whether such binding is an indication of sperm capacitation. Frozen–thawed semen samples from five bulls were assessed for antibody binding after 0, 2, 4 and 6 h of incubation. Maximal binding was observed at 4 h. Lysophosphatidylcholine (100 μg ml−1) induced acrosome reaction assay was performed to assess sperm capacitation at various intervals. A positive correlation observed between the degree of HS-11 binding and that of lysophosphatidylcholine-induced acrosome reaction suggested that HS-11 binds with capacitated, but not with acrosome-reacted, spermatozoa. Significant variation was observed among bulls in binding of HS-11 to spermatozoa. Split samples of frozen semen from the five bulls were also used for fertilization in vitro to assess their ability to fertilize and initiate cleavage of bovine oocytes matured in vitro. A close correlation (r = 0.90; P < 0.05) was observed between binding of HS-11 at 4 h and the cleavage rate of oocytes. The percentage increase in lysophosphatidylcholine-induced acrosome reaction was also correlated with HS-11 binding at 4 h (r = 0.81; P < 0.10). These results suggest that HS-11 is a potential marker for assessing pre-fertilization and post-thaw membrane changes in bull spermatozoa.
R. Rajamahendran, J. D. Ambrose, and C. Y. G. Lee
C. S. Haley, G. J. Lee, M. Fordyce, G. Baxter, R. B. Land, and R. Webb
Summary. A high and a low response line in sheep were selected on the basis of the mean concentration of LH in 10-week-old Finn–Dorset ram lambs after an i.v. injection of 5 μg GnRH. After 8 male generations the mean LH response of the high line was more than 5-fold that of the low line and the heritability of the selected trait was estimated at 0·44 ± 0·015. Highly significant line differences in mean LH response to GnRH were also found in males at 20 weeks of age and females at 10 and 20 weeks of age and the genetic correlations between the four LH response traits appear to be close to unity. Large line differences in the mean FSH response to GnRH were also found in both males and females at 10 and 20 weeks of age. Selection had little effect on the physical characteristics of lambs. High-response line ewes entering their first breeding season at about 7 months of age showed oestrus earlier in the season and had higher ovulation rates and numbers of lambs born per ewe lambing than did low-response line ewes. In the second breeding season, at about 19 months of age, the only line difference was a higher ovulation rate early in the breeding season in high-line ewes. It is suggested that these changes may be mediated by a more rapid response in high-line ewes to increased GnRH stimulation at puberty or at the beginning of the breeding season.
Keywords: genetic selection; LH release; GnRH; sheep
D. G. Whittingham, Maureen Wood, J. Farrant, H. Lee, and J. A. Halsey
Summary. The effect of the rate of rewarming on the survival of 8-cell mouse embryos and blastocysts was examined. The samples were slowly cooled (0·3–0·6°C/min) in 1·5 m-DMSO to temperatures between −10 and −80°C before direct transfer to liquid nitrogen (−196°C). Embryos survived rapid thawing (275–500°C/min) only when slow cooling was terminated at relatively high subzero temperatures (−10 to −50°C). The highest levels of survival in vitro of rapidly thawed 8-cell embryos were obtained after transfer to −196°C from −35 and −40°C (72 to 88%) and of rapidly thawed blastocysts after transfer from −25 to −50°C (69 to 74%). By contrast, for embryos to survive slow thawing (8 to 20°C/min) slow cooling to lower subzero temperatures (−60°C and below) was required before transfer to −196°C. The results indicate that embryos transferred to − 196°C from high subzero temperatures contain sufficient intracellular ice to damage them during slow warming but to permit survival after rapid warming.
Survival of embryos after rapid dilution of DMSO at room temperature was similar to that after slow (stepwise) dilution at 0°C. There was no difference between the viability of rapidly and slowly thawed embryos after transfer to pseudopregnant foster mothers. It is concluded that the behaviour of mammalian embryos subjected to the stresses of freezing and thawing is similar to that of other mammalian cells. A simpler and quicker method for the preservation of mouse embryos is described.
J. R. McNeilly, M. Fordyce, R. B. Land, G. J. Lee, and R. Webb
Summary. Testis diameter and body weight were recorded from 6 to 76 weeks of age in ram lambs from two established lines selected for high (H) and low (L) testis size. While testis growth was greater in the H line up to 14 weeks of age (P <0·001), body weight was significantly lower, with the L line rams being 10 kg heavier by 76 weeks. There were no differences in plasma LH up to 20 weeks of age, but FSH concentrations were significantly lower at 14 and 20 weeks in the H line. Testosterone concentrations were not significantly higher in the H line from 6 to 20 weeks. In lambs castrated at birth, significantly higher FSH values were recorded from 6 to 20 weeks of age in the H line (P <0·001) whereas there was no difference in LH concentration at 6 and 10 weeks of age between the lines. At 14 and 20 weeks, however, the concentrations of LH were greater in the H than L line lambs (P <0·05). After hemicastration at 6 weeks of age, the rate of growth of the remaining testis in the L line lambs was significantly faster than in entire lambs of that line from 10 to 20 weeks (P <0·05 at 10 weeks to P <0·001 at 20 weeks). There was no difference in the rate of testis growth between the entire and hemicastrated lambs from the H line from 6 to 12 weeks of age.
It can be concluded that there is an underlying genetic difference in pituitary gland and/or hypothalamic activity in ram lambs from the two selected lines.
C. S. Haley, G. J. Lee, M. Ritchie, and R. B. Land
Summary. Selection based upon testicular diameter adjusted for body weight at 6, 10 and 14 weeks of age was used to produce two lines of sheep, with either high or low testicular size. Ten generations of selection were carried out and the estimate of the realized heritability of the selection criterion was 0·53 ± 0·01. There were significant positive correlated responses to selection for testicular diameter at 6, 10 and 14 weeks of age, but the correlated responses in body weight at these ages were negative. In mature females, there were significant negative correlated responses to selection in premating body weight in the 1st, 2nd and 3rd breeding season and in the day of the first oestrus in the 2nd breeding season. Litter size per ewe mated had a small positive correlated response to selection in the second breeding season. This latter response appeared to be due to a positive correlated response in fertility, ewes from the High-line having a significantly higher probability of conceiving to a single mating than those from the Low-line. There was no significant correlated response in ovulation rate or litter size per ewe lambing and the genetic correlation between these traits and the selection criterion is likely to be close to zero. This may be due to the adjustment for body weight used, but it is possible that, in any event, body weight in young rams may be a better predictor of female ovulation rate than testicular diameter. These results do not rule out the possibility that testicular size in rams older than those selected would provide a good predictor of genetic merit for female ovulation rate.
Keywords: genetic selection; testicular diameter; ovulation rate; litter size; sheep
X J Yin, H S Lee, Y H Lee, Y I Seo, S J Jeon, E G Choi, S J Cho, S G Cho, W Min, S K Kang, W S Hwang, and I K Kong
This work was undertaken in order to study the developmental competence of nuclear transfer (NT ) into cat embryos using fetal fibroblast and adult skin fibroblast cells as donor nuclei. Oocytes were recovered by mincing the ovaries in Hepes-buffered TCM199 and selecting the cumulus oocyte complexes (COCs) with compact cumulus cell mass and dark color. Homogenous ooplasm was cultured for maturation in TCM199+10% fetal bovine serum (FBS) for 12 h and used as a source of recipient cytoplast for exogenous somatic nuclei. In experiment 1, we evaluated the effect of donor cell type on the reconstruction and development of cloned embryos. Fusion, first cleavage and blastocyst developmental rate were not different between fetal fibroblasts and adult skin cells (71.2 vs 66.8; 71.0 vs 57.6; 4.0 vs 6.1% respectively; P < 0.05). In experiment 2, cloned embryos were surgically transferred into the oviducts of recipient queens. One of the seven recipient queens was delivered naturally of 2 healthy cloned cats and 1 stillborn from fetal fibroblast cells of male origin 65 days after embryo transfer. One of three recipient queens was delivered naturally of 1 healthy cloned cat from adult skin cells of female origin 65 days after embryo transfer. The cloned cats showed genotypes identical to the donor cell lines, indicating that adult somatic cells can be used for feline cloning.
C. G. Tsonis, Helen Quigg, V. W. K. Lee, Lorraine Leversha, A. O. Trounson, and J. K. Findlay
Summary. Inhibin activity was measured by bioassay in follicular fluid of 99 individual ovine follicles ranging from 1·4 to 6·8 mm diameter (used to calculate volume) and in various stages of atresia. Treatment of samples before assay with charcoal concentrations of > 1 mg/ml resulted in significant loss of inhibin activity.
The inhibin content of follicular fluid from individual follicles varied with follicular fluid volume but not with the degree of atresia, as assessed by morphological criteria. Inhibin concentration was not related to atresia, but was correlated with follicular fluid volume. However, aromatase activity in granulosa cells and oestradiol-17β concentration of follicular fluid, considered to be good indices of atresia, were highly correlated with both inhibin content and concentration in follicles ≥ 3·5 mm diameter.
Inhibin in ovine follicular fluid shows marked variation between follicles and it is suggested that this reflects a combination of the number and activity of granulosa cells within the follicle and the exit rate of inhibin from the follicle.