EMS115810 Vasectomy 1

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Lab Anim (NY). Author manuscript; available in PMC 2021 August 01.
Published in final edited form as:
Lab Anim (NY). 2021 February 01; 50(2): 49–52. doi:10.1038/s41684-020-00692-w.
Europe PMC Funders Author Manuscripts
Replacement of surgical vasectomy through the use of wild-type

sterile hybrids
Chris Preece, Samy Alghadban, Amine Bouchareb, Daniela Moralli, Daniel Biggs, Benjamin
Davies
Wellcome Centre for Human Genetics, University of Oxford, Oxford, UK
Abstract
For the production and rederivation of mouse strains, pseudopregnant female mice are used for
embryo transfer and serve as surrogate mothers to support embryo development to term.
Vasectomized males are commonly used to render pseudopregnancy in females, generated by
surgical procedures associated with considerable pain and discomfort. Genetically modified mouse
strains with a sterility phenotype provide a non-surgical replacement, and represent an important
application of the 3Rs. However, the maintenance of such genetically modified mouse strains
requires extensive breeding and genotyping procedures, which are regulated procedures under
national legislation. As an alternative, we have explored the use of sterile male hybrids which
result when two wild-type mouse subspecies, Mus musculus musculus and Mus musculus
domesticus, interbreed. We find the male STUSB6F1 hybrid, resulting from the mating of female
STUS/Fore with male C57BL/6J, ideally suited and demonstrate that its performance for the
production of oviduct and uterine transfer recipients is indistinguishable when compared to
surgically vasectomized mice. The use of these sterile hybrids avoids the necessity for surgical
procedures or the breeding of sterile genetically modified lines and can be generated by the simple
mating of two wild-type laboratory strains - a non-regulated procedure. Furthermore, in contrast
with the breeding of genetically sterile mice, all male offspring are sterile and suitable for the
generation of pseudopregnancy, allowing their efficient production with minimal breeding pairs.
Introduction
For the production and rederivation of genetically modified (GM) mouse strains,
pseudopregnant recipient females for embryo transfer are required. Female recipients are
conventionally rendered pseudopregnant by mating with vasectomized males, prepared by
surgical bisection of the vas deferentia1. Outbred mice and certain hybrids of commonly
Correspondence to: Benjamin Davies.

ben. davies@well.ox.ac.uk.
Reporting Summary.
Further information on research design is available in the Nature Research Reporting Summary linked to this article
Author contributions
C.P. and S.A. contributed equally to this work. C.P., D.B. and S.A. performed the animal work; D.M. performed the cytological
analysis of spermatocytes; S.A. and B.D. designed the study; S.A. and A.B. analysed the data and B.D. wrote the manuscript.
Competing interests
The authors declare no competing interests.
Preece et al. Page 2
available inbred strains are frequently used for this purpose, due to their robust fecundity.
The vasectomy procedure is performed surgically under general anaesthetic and is
considered a procedure of moderate severity. Over the years, the procedure has been refined,
with many groups now performing the procedure via scrotal sac incision, rather than midline
laparotomy2. Despite this refinement, there is little evidence that this refinement alleviates
any of the pain, discomfort and distress which inevitably accompanies this surgical
intervention3.
GM mice with a phenotype of male sterility can be used to avoid this surgical procedure
entirely and have been welcomed by the community. Both knock-out and transgenic mice
have been used for this purpose. For example, homozygous knock-out males lacking the
Gapdhs gene4 and the transgenic model, Tg(Prm1-EGFP)#Ltku5, have been used
successfully for the production of pseudopregnant recipient females. Although the use of
GM strains clearly replaces the surgical procedures, the production of these strains
necessitates breeding and genotyping procedures, both regulated procedures under national
legislation. Indeed, for knock-out mouse strains, homozygous male mice are required and
only 12.5% of the mice generated from heterozygous crosses would be of the correct
genotype and gender. For transgenic strains, only 25% of the mice generated from breeding
hemizygous transgenic females can be used. Subsequently, the breeding of these genetically
sterile mice incurs a considerable animal cost. Furthermore, transgene silencing is a well-
recognized phenomenon that occurs in conventionally generated transgenic models6, and
thus a risk of silencing and restoration of fertility exists when using such models.
Consequently, many groups using transgenic sterile lines perform testing of studs to ensure
complete sterility before relying on individual males. These fertility checks add an
additional burden of breeding procedures when using such GM models.
In many species, hybrids of closely related strains and species show a sterility phenotype; for
example mules are sterile offspring of horses and donkeys7. This phenomenon is widely
considered to be the first step in the process of speciation. Where two populations become
geographically isolated and start to diverge into two distinct species, when the circumstances
permit that the two populations re-encounter each other and the divergence still permits
breeding, the male offspring of such inter-specific breeding are frequently sterile. The
phenomenon was reported in the 1920s and is known as Haldane’s rule8.
In mouse, there are a number of cases of hybrid sterility reported and models have been
established in the laboratory. When laboratory mice, e.g. C57BL/6J Mus musculus
domesticus, are bred with wild-caught Mus musculus musculus strains of mice, e.g.
PWD/PhJ or STUS/Fore, the fertility of the resulting male offspring is frequently
impaired9, 10. When using PWD/PhJ, the direction of the cross is important, due to a
modifier locus on the X-chromosome11, 12. Male PWDB6F1 hybrids, generated from crosses
between male C57BL/6J and female PWD/PhJ mice, are completely sterile, whereas male
B6PWDF1 hybrids, generated from crosses between male PWD/PhJ and female C57BL/6J
mice are subfertile13, 14. Interestingly, hybrids between C57BL/6J and STUS/Fore parental
strains were reported as being sterile in both directions14.
Given the simplicity of producing sterile males by interbreeding wild-type subspecies of

laboratory mouse, in this study we explore the possibility of using natural sterile hybrids for
the production of pseudopregnant females for embryo transfer.
Results
Selection of appropriate strains for the production of sterile hybrid males

Given that hybrids between C57BL/6J and STUS/Fore mice had been reported as being
sterile in both cross directions, we initially chose to facilitate the production of sterile
hybrids by using male STUS/Fore mice bred with C57BL/6J females. In test breeding of the
resulting B6STUSF1 hybrid males with 8-week old CD1 females, however, we noticed a
low level of litter production from these B6STUSF1 hybrids when mated at 12-18 weeks of
age, with two of 11 B6STUSF1 hybrids tested yielding litters, indicating that the hybrid
generated in this direction, was in fact subfertile. This subfertility was confirmed by
investigating the caudal epididymis of B6STUSF1 males for the presence of mature
spermatozoa, which were found albeit in low concentrations in comparison with age
matched C57BL/6J mice (Fig. 1a).
As a result, we verified the fertility of STUSB6F1 hybrid mice generated from crosses of
male C57BL/6J with female STUS/Fore mice and confirmed that these mice were
completely sterile, as previously reported14. Test breeding of 8 STUSB6F1 hybrids with
CD1 females resulted in no litters, and absolute sterility was confirmed by a lack of caudal
epididymal sperm (Fig. 1a) and sperm production in seminiferous tubules (Fig. 1b).
Furthermore, investigation of spermatocytes from STUSB6F1 males revealed abnormal
chromosome pairing with a persistence of unrepaired intermediates and failure to form a
distinct sex body, as revealed by γH2AX staining (Fig. 1c, d). STUSB6F1 hybrid males
were thus selected for this study.
Performance of STUSB6F1 sterile males in rendering pseudopregnancy

STUSB6F1 hybrid males between 10 and 30 weeks of age were mated with CD1 females
between 8 and 16 weeks of age over the course of several months, to prepare embryo
transfer recipients for both oviduct and uterine embryo transfer. To establish the utility of the
sterile hybrids, the plugging rate of CD1 females was measured and compared to
contemporaneous data from the laboratory using conventionally prepared surgically
vasectomized CD1 mice (between 10 and 30 weeks of age). There was no significance
difference in plugging rate between these groups (Table 1, X2=0.124 P=0.72), suggesting
that the hybrid males were entirely suitable for the production of pseudopregnant embryo
transfer recipients.
The plugged CD1 females were used for embryo transfers using 2-cell embryos surgically
transferred to the oviduct at 0.5 dpc, or blastocyst stage embryos surgically transferred to the
uterus at 2.5 dpc. The embryos used were all of the same strain, C57BL/6J, and were either
unmanipulated, microinjected or electroporated. To avoid any impact of the manipulation,
experiments where there was any known or assumed consequence of the genetic
modification on embryonic development were excluded from the analysis. We were unable
to detect any significant difference in pregnancy rate for both oviduct embryo transfer (Table
1, X2=0.139; P=0.71) and uterine embryo transfer (Table 1, X2=1.388; P=0.24), when
comparing the pseudopregnant recipient females prepared with the naturally sterile hybrids
with those prepared with surgically vasectomized CD1 mice. These results again suggest
that the hybrid males could provide a non-surgical alternative for the preparation of embryo
transfer recipients.
To confirm that the CD1 mice rendered pseudopregnant using the sterile hybrid males were
entirely normal in their ability to raise litters, the birth rate expressed as the number of live
born pups as a percentage of embryos transferred, was measured for each pregnant embryo
transfer and compared with that of CD1 recipients conventionally prepared using surgically
vasectomized CD1 mice. The birth rate resulting from oviduct embryo transfer and uterus
embryo transfer showed a wide distribution of efficiencies, as expected for real project data.
There was, however, no impact of how the recipient females was prepared (Fig. 2a, b)
(oviduct transfer: X2=0.848; P=0.36; uterus transfer: X2=0.021; P=0.89). Given that an
approximately constant number of embryos were transferred per pseudopregnant female
(oviduct: 26.9±4.6; uterus: 19.3±2.3) an assessment of the distribution of the litter size was
meaningful and this too was not impacted by the method used to render the recipient female
pseudopregnant (Fig. 2c, d) (oviduct transfer: Mann-Whitney test: P = 0.65; uterus transfer:
Mann-Whitney test: P = 0.42).
In summary, the sterile wild-type hybrid STUSB6F1 male mice performed indistinguishably
from conventionally surgically vasectomized male mice in their ability to render CD1 mice
pseudopregnant for the purpose of embryo transfer. No deviation in plugging ability,
pregnancy rate nor live birth rate was observed.
Performance of STUSB6F1 females as embryo transfer recipients

Given that the male hybrids can be efficiently generated in the laboratory by interbreeding
two wild-type strains, the females resulting from this production would be superfluous. To
avoid this animal wastage, we were keen to explore whether the female STUSB6F1 hybrids
could themselves serve as recipient females, especially since hybrid females are often
considered to be very robust and good mothers. Subsequently, we performed a pilot study to
assess pregnancy rate and birth rate following oviduct embryo transfer of 2-cell embryos
into plugged 0.5 dpc STUSB6F1 females and compared these figures against
contemporaneous transfers into plugged 0.5 dpc CD1 females. Of 14 oviduct transfers
performed, 8 became pregnant and gave birth to live offspring, representing a pregnancy rate
of 57%, which was less than the 71% pregnancy rate observed using CD1 mice at this time
period. Although not statistically significant due to the small size of the pilot study
(X2=0.525, P = 0.47), this data did raise concerns about the suitability of this strain. Indeed,
the STUSB6F1 females were found to be significantly smaller in size and leaner than aged
matched CD1 females, making aspects of the surgery more challenging, for example
exposing and restraining the reproductive organs via the ovary fat pad was particularly
challenging in STUSB6F1 females. This could well explain the reduction in pregnancy
levels seen when using hybrid females. The live birth rate obtained from these pregnant
transfers was more evenly matched (STUSB6F1: 16.3%, CD1: 14.2%) between STUSB6F1
and CD1 females, with each foster being transferred bilaterally with approximately constant
numbers of 2-cell embryos (STUSB6F1: 22.6±4.6; CD1: 24.5±4.5). The results of this pilot
experiment suggest that the STUSB6F1 could act as a suitable embryo transfer recipient
strain but the technical aspect of performing surgery on leaner mice and the lower observed
incidence of pregnancy demand further investigation.
Discussion
We have explored the use of naturally sterile hybrid mice for the generation of
pseudopregnant embryo transfer recipients and have found STUSB6F1 males to be a
suitable replacement for surgical vasectomized males. Importantly, both plugging rate and
pregnancy rate following both oviduct and uterine embryo transfer were unaffected by the
choice of male used to render the recipient female pseudopregnant. Furthermore, the average
litter size was unaffected by the use of sterile hybrid mice.
The use of such hybrids obviates the need for a surgical procedure and thus can be
considered an impactful “Refinement” with respect to the 3Rs. When compared against
phenotypically sterile GM strains, the production of the naturally sterile hybrid is
considerably more efficient, since 50% of all the mice produced (i.e. all males) are sterile
and suitable for use for the preparation of pseudopregnant embryo transfer recipients. In
contrast, for the breeding of phenotypically sterile GM male mice only 12.5%-25% of the
production can be used. Thus, the use of sterile hybrids constitutes an important 3Rs
“Reduction” when compared with the use of GM strains. Furthermore, the breeding of these
naturally sterile hybrid mice involves the simple pairing of two wild-type strains which, in
contrast to the maintenance and breeding of GM strains, does not constitute a regulated
procedure under national legislation, with no biopsy and genotyping procedures needed.
To maximize the 3Rs impact, we had considered that the female hybrid mice produced as
litter mates could be used as embryo transfer recipients. Our pilot study suggested that
whilst embryo transfers into this strain could be successful with the average pup production
rate unaffected, the number of STUSB6F1 hybrid mice becoming pregnant following
embryo transfer was lower than that observed using outbred CD1 mice. Part of the reason
for this decrease in efficiency was thought to be the increased technical difficulty of
performing surgery on the physically smaller and leaner hybrid. One future area of research
would be to assess whether these females could be suitable for non-surgical embryo transfer
of blastocysts, since this would obviate the need for any surgical procedure. Despite the
potential for a further 3Rs Reduction that the use of the hybrid females as embryo transfer
recipients could convey, the small scale of the colony required for the production of sterile
males would most probably not satisfy the demands of the average production facility for
recipient females.
The main disadvantage of the use of naturally sterile hybrids, is the need to maintain the two
parental lines. Whereas the Mus musculus domesticus strain C57BL/6J is widely available
and can be purchased readily from a number of commercial suppliers, Mus musculus
musculus strains such as STUS/Fore, or PWD/PhJ are not readily commercially available
and subsequently an additional colony must be maintained, which clearly involves breeding
procedures, albeit non-regulated ones. Initially we had hoped to rely upon the B6STUSF1
hybrid, which had been reported as being sterile14. This strain could be generated by IVF of
C57BL/6J embryos using thawed frozen sperm from this Mus musculus musculus strain,
obviating the need to maintain a live colony of this substrain. However, this B6STUSF1
hybrid displayed a low level of fertility and two hybrids, at 15 and 18 weeks of age
respectively, were able to sire litters. Although this observation is in disagreement with a
previous report that tested the fertility of the B6STUSF1 between 8-12 weeks of age14, a
recent publication has highlighted age-dependent increases in fertility in hybrid strains of
similar subspecies, starting at 15 weeks of age15, which we believe explains this
discrepancy. Since it is the females of the Mus musculus musculus strain that need to be
used as mothers for the production of the fully sterile STUSB6F1 hybrid, on account of the
modifying locus on the X-chromosome, the production necessitates a breeding colony of
STUS/Fore mice.
We have found the maintenance of the STUS/Fore mouse strain to be more challenging than
that of standard laboratory strains since the reproductive attributes of this strain are not ideal
for large scale laboratory maintenance, with small litter size observed (Table 2). Potentially
this is simply a reflection of in-breeding depression, since this line was imported in small
numbers. Indeed, when STUS females are used for the production of the sterile hybrid
offspring, their litter size increases to acceptable ranges (Table 2). The alternative Mus
musculus musculus strain, PWD/PhJ, may provide an alternative as, in our hands, the
reproductive characteristics were more robust (Table 2) and the strain is freely available
from repositories (e.g. Jackson Laboratory Stock No: 004660). PWD/PhJ was not used in
this study simply because of their small size which can present husbandry problems when
mated with C57BL/6J mice, but most importantly because of their erratic temperament.
Despite the inherent husbandry difficulties that maintenance of a Mus musculus musculus
entails, the 3Rs advantages of “Refinement” with respect to the use of surgical vasectomized
mice and the “Reduction” with respect to the use of phenotypically sterile GM mice,
outweigh these disadvantages.
Methods
Animal experiments
Wild-type STUS/Fore mice were obtained from the laboratory of Jaroslav Piálek, Institute of
Vertebrate Biology, Brno and wild-type C57BL/6J and CD1 mice were obtained from
Charles River Laboratories, UK. B6STUSF1 hybrid mice were produced by mating
C57BL/6J females with STUS/Fore males, and STUSB6F1 hybrid mice were produced by
mating STUS/Fore females with C57BL/6J males. Examples of the parental strains and the
STUSB6F1 hybrid mice are shown in Supplementary Figure 1.
Mice were housed in individually ventilated cages in 12:12 hr light cycle, with access to
food and water ad libitum. All studies were conducted with institutional ethical review panel
approval and in accordance with the UK Home Office Animals (Scientific Procedures) Act
1986 under license PAA2AAE49. A minimum of 60 embryo transfers per experimental
group were performed to ensure sufficient power for detecting a significant change in
success rate. Embryos were randomly allocated to experimental group on the basis of
availability of plugged females. Due to the difference in colour of the mice (albino CD1
versus agouti STUSB6F1), the surgical procedures could not be carried out with blinding.
Sterility assessment
Sterility of F1 males was assessed from 12 weeks of age by mating with sexually mature (8
weeks old) CD1 females. The females were checked daily for the presence of vaginal plugs
and, when observed, the females were kept for three weeks to confirm pregnancy status.
Hybrid males were culled between 15 and 25 weeks of age and sperm counts was obtained
by dissection of caudal epididymis and dispersal of the sperm in 1000 μl of warm PBS prior
to counting with a haemocytometer.
Assessment of normal sex body formation

Mouse testis chromosome spreads were prepared and immunostained as previously
described9. The following primary antibodies were used: mouse anti-SYCP3 (Santa Cruz
Biotechnology sc-74569, D-1) and chicken anti-phospho-H2AX (Biorbyt, orb195374) and
Alexa Fluor 488- or 594-conjugated secondary antibodies against chicken IgY and mouse
IgG respectively (ThermoFisher Scientific). The slides were mounted in DAPI/Vectashield
(Oncor) and analysed with an Olympus BX60 microscope for epifluorescence, equipped
with a Sensys CCD camera (Photometrics, USA), using Genus Cytovision software (Leica).
A minimum of fifty pachytene cells per sample were analysed and the normal appearance of
the sex bodies was assessed based on the staining pattern.
Surgical vasectomy
Male CD1 mice at 5 weeks of age were surgically vasectomized using the scrotal incision
technique2, 16 and left to recover for at least 3 weeks prior to use. All vasectomized mice
were test-bred with 8-week-old CD1 females to ensure sterility prior to use in embryo
transfer experiments.
Surgical embryo transfer

Mature CD1 (8 to 16 week old) females were mated with either vasectomised CD1 males or
sterile STUSB6F1 males (10 to 30 weeks of age) to induce pseudopregnancy and copulation
was confirmed by the presence of vaginal plugs the next morning. Embryos for oviduct
transfer were either non-manipulated for the purpose of rederivation (29% of transfers) or
resulted from genetic manipulation by either electroporation of CRISPR reagents17 (34% of
projects) or microinjection18 of either CRISPR reagents (19% of projects) or DNA
constructs (18% of projects), following by culture in vitro to the 2-cell stage. Embryos for
blastocyst transfer resulted from ES cell microinjection and were left to recover in vitro for
several hours prior to transfer. Surgical transfer to pseudopregnant females was performed
bilaterally into either oviduct (2-cell embryos) or uteri (blastocyst) using standard
techniques1. For the pilot study for surgical transfer into STUSB6F1 females, STUSB6F1
females of 8-12 weeks of age were prepared by mating with sterile STUSB6F1 males (10 to
30 weeks of age) and were transferred with electroporated 2-cell embryos, bilaterally into
the oviducts. Plugging rate of individual mice was recorded as well as pregnancy rate and
the litter sizes generated from pregnant females.
Statistical analysis
Data sets were analysed and presented using Graphpad Prism software v8.4, using
measurements from distinct samples. Differences between groups were assessed using a
Chi-squared test for comparison of proportions (for plugging, pregnancy and birth rates) and
Mann Whitney tests for analysis of litter size.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgements
This work was supported by a Wellcome Trust Core Award Grant [203141/Z/16/Z] and a National Centre for the
Replacement, Refinement and Reduction of Animals in Research project grant [NC/R001014/1].
Data availability
All data generated or analysed during this study are included in this published article (and its
Supporting Information files). The STUS/Fore strain is available on request.
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Fig. 1. Confirmation of the infertility of STUSB6F1.

a, Box plot showing the range of total sperm count for subfertile B6STUSF1 (n=11),
infertile STUSB6F1 (n=8) and fertile C57BL/6J mice (n=4). b, Haematoxylin and eosin
section through the testes of STUSB6F1 (left panel) and C57BL/6J (right panel). c,
Incidence of normal sex body formation in spermatocyte spreads of B6STUSF1 (n=2),
STUSB6F1 (n=3) and C57BL/6J mice (n=6) (% are determined from at least 50 pachytene
stages cells). Error bars show standard deviation. d, Example immunostaining of γH2AX
(red) and SYCP3 (green) showing unrepaired chromosome intermediates in STUSB6F1
mice (left panel) and normal localization of the γH2AX marker to the sex body in fertile
C57BL/6J mice (right panel).
Fig.2. Comparison of litter production.

a, Production efficiency as measured by live pups born per embryos transferred bilaterally
into oviducts of embryo transfer recipients rendered pseudopregnant with either STUSB6F1
hybrid (F1; n=122) or surgically vasectomized (Vas; n=136) studs. The colours indicate the
type of embryo manipulation performed (DNA microinjection (red), CRISPR microinjection
(blue), CRISPR electroporation (grey) and non-manipulated (green). b, Production
efficiency as measured by live pups born per microinjected blastocysts transferred bilaterally
into uteri of embryo transfer recipients rendered pseudopregnant with either STUSB6F1
hybrid (F1; n=61) or surgically vasectomized (Vas; n=74) studs. For a, and b, all data points
are shown and the means and the standard deviation of the collected data are indicated. c-d,
Mean litter size generated from bilateral embryo transfer recipients rendered pseudopregnant
with either STUSB6F1 hybrid (F1) or surgically vasectomized (Vas) studs for c, oviduct and
d, uterus) transfer. Error bars show the standard deviation.
Table 1
Comparison of STUSB6F1 hybrid versus surgically vasectomized studs on plugging
ability using non-oestrus checked CD1 females and pregnancy rate following oviduct and
uterus transfer.
Pregnancy rate Pregnancy rate

Strain Plugging rate
(oviduct transfer) (uterus transfer)
34.5 % 83.1% 69.2 %
Vasectomized CD1
(n=641) (n=154) (n=107)
33.3 % 85.3% 60.4%

STUSB6F1 n.s. n.s. n.s.
(n=445) (n=163) (n=101)
n.s. – non significant difference (P>0.05).

Table 2
Reproductive parameters of parental and hybrid colonies
Colony Name Number of litters assessed Average Litter Size

STUS/Fore 84 3.7
STUSB6F1 30 4.9
B6STUSF1 11 7.5
PWD 98 5.1
C57BL6/J 304 6.1

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Europe PMC Funders Group

Replacement of surgical vasectomy through the use of wild-type

Correspondence to: Benjamin Davies.

Given the simplicity of producing sterile males by interbreeding wild-type subspecies of

Selection of appropriate strains for the production of sterile hybrid males

Performance of STUSB6F1 sterile males in rendering pseudopregnancy

Performance of STUSB6F1 females as embryo transfer recipients

Assessment of normal sex body formation

Surgical embryo transfer

Fig. 1. Confirmation of the infertility of STUSB6F1.

Fig.2. Comparison of litter production.

Pregnancy rate Pregnancy rate

33.3 % 85.3% 60.4%

n.s. – non significant difference (P>0.05).

Colony Name Number of litters assessed Average Litter Size

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