The grey squirrel is an invasive alien species in the UK. The economic impact of grey squirrels on England and Wales' forestry is estimated to be around £37 million per year, and the presence of this species has caused the decline of the native red squirrel due to interspecific competition and transmission of diseases. Oral contraceptives, delivered in baits, are being developed to decrease the number of grey squirrels. Bait uptake can be affected by many factors, which must be understood to maximise the effects of oral contraceptives on grey squirrel populations. This study investigated the effect of sex, age, and reproductive status on bait uptake by grey squirrels. Squirrels were pit-tagged and bait uptake measured using squirrel-specific bait dispensers (hoppers) fitted with pit-tag readers and baited with hazelnut paste and the biomarker Rhodamine B for 5 days. For each visit to the hoppers, the identity of the squirrel, date, time, duration, and ID of the hopper visited were recorded. One to 3 weeks later, as many squirrels as possible were recaptured and assigned categories related to sex, age (immature, adult), and reproductive status (reproductively active or non-active). The presence of Rhodamine B in the hair was also recorded in all these animals as an indicator of bait uptake. Overall, 49 % of squirrels were positive for Rhodamine B, with sexually active females showing the highest bait uptake. Adult squirrels made fewer daily visits to hoppers and visited fewer hoppers per day than immature squirrels, though both of these parameters showed greater variability in immature animals than in adults. Sex, age, and reproductive status did not affect bait uptake in grey squirrels at the density of grey squirrels (3.9 – 6.3 squirrels/ha) and bait hoppers (3/ha) tested in this study. As several doses of an oral contraceptive are likely to be required to effectively suppress reproduction, this study confirmed that it should be possible for contraceptives to have an equal chance of affecting each category of squirrels within a relatively short period of time. As sexually mature females drive population growth, targeting these females with oral contraceptives should be a priority.  These results could be used to inform targeted interventions aimed at delivering baits containing contraceptives or vaccines to grey squirrels and for population management.

All field work was conducted by trained APHA operatives. Trapping was conducted over a 2-to-3-week period for 3-4 trap days per week per wood. Grey squirrels were captured using single-capture cage traps deployed at a density of 3/ha across each wood. Trapped squirrels were anaesthetised on site with isoflurane via a portable anaesthetic machine and a mask. Under anaesthesia, squirrels were health checked and equipped with a subcutaneous PIT-tag (Identichip®, York, UK) inserted in the scruff of the neck, as a unique identifier. Within 2 weeks of the last animal equipped with a PIT-tag, traps were removed and squirrel bait dispensers, hereafter referred to as hoppers, were deployed across each wood at a density of 3/ha (21-24 in total). A hopper consisted of a bait compartment within a 10 × 10 × 55 cm length of aluminium tubing and a door that squirrels had to lift to access the bait. Each hopper was equipped with a PIT-tag reader to record the date and time that each tagged animal entered the hopper (Beatham et al. 2021). The hopper trial was divided into a pre-baiting and baiting phase. During a ‘prebaiting’ phase, lasting one week, hoppers were baited 3 times, every other day for the first 5 days, and the hopper door was left open to encourage the squirrels to use these devices. The bait consisted of 40 g of hazelnut paste per hopper, as in Beatham et al. (2021).

During the baiting phase, which started the following week, each hopper was set so that squirrels had to lift the door to access the bait. Data collection on squirrel visits to hoppers began during the baiting phase and lasted 5 days. During the baiting phase, 0.18% (weight/weight), Rhodamine B (CAS number 81-88-9, Sigma-Aldrich) bait marker was added to the hazelnut paste to monitor population bait uptake. Within 4 weeks after the end of the baiting phase, a trap and remove operation was carried out in each wood to recapture as many squirrels as possible. All trapped squirrels were humanely dispatched according to Schedule 1 of the Animals (Scientific Procedures) Act 1986 (UK) and scanned to assess whether they had a PIT-tag. Hair samples were collected from the flank and analysed using an Ultra-Violet microscope, to test for Rhodamine B presence (Fisher 1973, Beatham et al. 2024). The carcasses were placed in a -20 ^oC freezer until post-mortem examinations.

During post-mortem examinations, squirrels were assigned a sex, age, and reproductive status. The age of each squirrel was assessed through tooth eruption, replacement, and wear following the method by Hench et al. (1984). Based on this method, 4 age classes were identified: A) < 6 months; B) between 6 months and 14 months; C1) between 14 and 36 months; C2) > 36 months. For female squirrels, reproductive status was assessed based on a combination of features of the vulva and uterus. Vulva scores were defined as: 1 = width < 5mm, no noticeable swelling, pale pink; 2 = width 5mm – 7mm, some swelling, pink; 3 = > 7mm, very swollen, darker pink. Uterus status was defined as small, ribbon-like, pale pin, or oedematous, or oedematous with embryonic vesicles, embryos, or foetuses (Maranesi et al. 2020). Uterine horn (width measured as a cross-section of the middle point of a uterine horn) was defined as non-reproductively active if ≤ 2.5 mm, or reproductively active if > 2.5 mm.  The vesicles, also referred to as “vitelline vesicles,” are the first structures that can be observed inside the gestational sac; they have a roundish cystic shape with a fine wall (Luis Alcázar and Alcázar, 2008). Based on a combination of the above variables, females were assigned to the following categories:

1 - Immature - The squirrel has not reached sexual maturity. The animal presents tooth wear A/B, vulva score 1-2, uterus horn width < 2.5 mm, ribbon-like and pale pink uterus.

2 - Adult, not reproductively active - The animal presents tooth wear C1/C2, vulva score 1-2, uterus horn width ≤ 2.5 mm, uterus is small, dark-pink and ribbon-like, and absence of oedema.

 3 - Adult, reproductively active - The animal presents tooth wear C1/C2, vulva score 1-2-3, the uterus is round, oedematous, and blood-red, characteristic of a sexually active animal. Sometimes vesicles, embryos, and foetuses show ongoing pregnancy. Uterus horn width >2.5 mm.

For male squirrels, reproductive status was assessed based on the method developed by Ferryman et al. (2006) as follows: testicle position (abdominal/scrotal); scrotal area colour (pink, light grey, dark grey, or black); scrotal and periscretal hair/stain; external paired testicle length. These 4 variables were used to determine the Visual Breeding Score (VBS, Figure 2). Based on the above variables, males were assigned to the following categories:

 1 – Immature - The squirrel has not reached sexual maturity. The animal presents tooth wear A/B. Testes can be abdominal or scrotal. Length of testicles (when scrotal) is approximately 1 cm, and colour is usually pink/light grey and completely covered in fur. The VBS value is 0/1/2.

2 – Adult, not reproductively active - Male squirrels generally have a period of sexual quiescence in summer and sexual redevelopment (recrudescence) in autumn. If recrudescence is not prolonged as a single peak of sexual activity, males become sexually active again in winter until May (Dubock 1979). The recrudescence and quiescence are characterised by low sperm production with a reduced testicular size. During this period, squirrels’ testicles are scrotal or abdominal, the colour of the scrotum is dark with a grey tone, the length of the external testicles is between 0 cm and 4.4 cm, and the presence or absence of scrotal hair. The animal presents tooth wear C1/C2. The VBS value is 2-5.

3 - Adult, reproductively active - Testicles are scrotal, the scrotum is dark in colour with a dark grey/black hue, and scrotal hair is absent. The VBS value is ≥ 6, confirming a progressive increase in the concentration of spermatozoa and reproductive capacity, and the length of the testicles is between 2.3 cm and 6.5 cm (Ferryman et al. 2006).

Statistical analyses

Analyses were carried out on 2 datasets. The first dataset (squirrel_nimble_rhod.csv) contained the records of age, gender, and reproductive status of each squirrel and whether it tested positive for Rhodamine B. The second dataset (squirrel_nimble_time.csv) contained the age, gender, and reproductive status of each squirrel and the hopper PIT-tag records associated with that squirrel. PIT-tag records were aggregated into 12-hour periods over the course of 5 days, resulting in 8 periods (with recording starting at 12.00 on the first day and ending at 12.00 on the fifth day).

The number of hoppers visited, the number of feeding visits, and the total time spent at any hopper were calculated for each squirrel. A feeding visit was defined as all PIT-tagged animals detected within two consecutive minutes for the same squirrel by the same hopper. A two-minute interval was used to standardise feeding visits, as squirrels would frequently move in and out of hoppers within one visit, and the hoppers used in different years had different levels of sensitivity regarding PIT-tag detection. The total time spent at any hopper was calculated from the time of the first PIT-tag record until the time of the last PIT-tag record for that visit. In instances where a squirrel was detected at the hopper only once, so the duration could not be calculated, a minimum time of 1 second was assigned.