Data extraction.

Following full text review, key information was extracted from each of the 33 retained publications using a standardised form (see S3 File) and tabulated. Study characteristics extracted and charted were: author, publication year, data source or country, study design, sample size, study aims, details of the intervention, health outcomes, how outcomes were measured and whether dogs were randomly or retrospectively allocated to study groups. Study population characteristics extracted and charted were: how the sample was sourced, dog breed, age and sex, whether dogs neutered for a related health problem were excluded, whether previous breeding history was considered and how puberty was defined. This was carried out independently by one researcher.

Summarising the available evidence.

The 33 publications were assessed by two researchers. Significant effects of timing of neutering on each health outcome were reported for either bitches or dogs and bitches (where studies did not present results for sexes of dogs separately). Studies that found no significant effects, reported significant effects only in male dogs, or made no comparisons between different neuter age groups were not included in the charting tables. A summary column was included to show the reported impact of neutering at certain times more clearly, including direction of any effect, for each health outcome included from each study.

Atopy.

The primary literature search identified 50 publications following the removal of duplicates. Thirty-five were excluded on title review, seven were excluded following abstract review and seven were excluded on full-text review, including six for concept and one that was not available in English. Therefore, one publication remained for assessment (Fig 1A).

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Fig 1.

a. The number of publications that were identified, reviewed, and excluded from the scoping review on the effect of neutering timing on atopy in female dogs. b. The number of publications that were identified, reviewed, and excluded from the scoping review on the effect of neutering timing on developmental orthopaedic disease in female dogs. c. The number of publications that were identified, reviewed, and excluded from the scoping review on the effect of neutering timing on neoplasia in female dogs. d. The number of publications that were identified, reviewed, and excluded from the scoping review on the effect of neutering timing on obesity in female dogs. e. The number of publications that were identified, reviewed, and excluded from the scoping review on the effect of neutering timing on urogenital disease in female dogs.

https://doi.org/10.1371/journal.pone.0311779.g001

Developmental orthopaedic disease.

The primary literature search identified 74 publications following the removal of duplicates. Twenty-eight were excluded on title review, 29 were excluded following abstract review and four were excluded on full-text review, including one that was not available in English, one that did not have full text available, one for concept and one for study design. Therefore, 13 publications remained for assessment (Fig 1B).

Neoplasia.

The primary literature search identified 513 publications following the removal of duplicates. Three hundred and forty were excluded on title review, 137 were excluded following abstract review and 25 were excluded on full-text review, including seven that were not available in English, three that did not have full text available, five for publication type and 10 for concept. Therefore, 11 publications remained for assessment (Fig 1c).

Obesity.

The primary literature search identified 182 publications following the removal of duplicates. One hundred and seven were excluded on title review, 57 were excluded following abstract review and 12 were excluded on full-text review including one that was not available in English, two for publication type and nine for concept. Therefore, six publications remained for assessment (Fig 1D).

Urogenital.

The primary literature search identified 326 publications following the removal of duplicates. Two hundred and twenty-five were excluded on title review, 63 were excluded following abstract review and 20 were excluded on full-text review, including two that were not available in English, two that did not have full text available, six for publication type and 10 for concept. Therefore, 18 publications remained for assessment (Fig 1E).

Neoplasia.

Only one of the 11 studies that investigated neoplasia considered pubertal status at neutering [20]. In that case-control study malignant mammary neoplasia was less likely in bitches neutered before experiencing any oestrus (relative risk 0.005) compared to those that had experienced one oestrus before neutering (relative risk 0.08) and two or more oestrous cycles before neutering (relative risk 0.26). However, as reported by Beauvais et al. [1], there is a risk of bias due to recruitment of cases and controls from different time periods and controlling for confounding variables in analysis.

The remaining 10 studies investigated the effects of timing of neutering related to age. Six of these made no comparisons of disease incidence between age at neuter groups, only between each age at neuter group and a group of entire dogs [14–17, 21, 60]. Four studies did make comparisons and one of those reported no impact of neutering age [12].

Three studies did report effects of neutering age, specifically on mammary tumours [45]; appendicular bone sarcoma [48] and HSA and MCT [13]. Beaudu-Lange et al. [45] reported a protective effect of neutering before two years of age on mammary tumour development. While this is similar to the findings of Schneider et al. [20], the study included a relatively small number of bitches (n = 50) that were neutered <2 years of age compared to those neutered >2 years (n = 243). Forty-seven of the bitches neutered >2 years of age were known to have a mammary tumour at the time of neutering and do not appear to have been excluded from the analysis. Torres de la Riva et al. [13] also reported more favourable outcomes for earlier neutering for HSA and MCT for 242 female Golden Retrievers. In that study, bitches were grouped as those neutered before (n = 172) or at/over 12 months of age (n = 70). Data were gathered from veterinary hospital records which could affect how well they represent the general dog population, however, confirmation of diagnosis was reliable. Bitches were studied to nine years of age, which could impact disease occurrence reporting. Additionally, there was no difference between bitches neutered before or at/after 12 months of age in the incidence of LSA although, in contrast to other types of neoplasia, a higher percentage of bitches in the early (5.9%) rather than late neuter group (1.4%) were diagnosed. Bitches with disease prior to neutering were excluded, the study only included one breed and sex, addressing the potential for these confounding variables to impact the results.

In contrast to the first two studies, Cooley et al. [48] found an increased risk with younger age at neuter for bone sarcoma in 683 Rottweilers, 389 of which were female. Data for neuter age were considered as months sexually entire on a continuous scale and were also grouped into neuter age groups and were reported for males and females separately. While data were gathered from an owner questionnaire, completed with veterinarian assistance, diagnoses were confirmed by reviewing radiographs which reduced the risk of incorrect diagnoses. Data on dog height and weight were included as confounding variables. It is not clear whether there were any dogs with bone sarcoma diagnosed before neutering and no mention of whether such cases were excluded from analysis.

Obesity.

None of the six studies that investigated obesity considered timing of neutering by pubertal status; all investigated effects of neutering timing related to age. One of the studies did not compare disease incidence between neuter age groups, only between each neuter age group and a group of entire dogs [55]. Five studies did make comparisons and three of those reported no impact of neutering age on overweight/obesity [10, 11, 50].

Two studies reported effects of age at neutering on obesity with contrasting results [12, 18]. Both studies considered being overweight and obese rather than obesity alone. Spain et al. [12] reported a decreasing risk of overweight body condition with decreasing age at neuter for 1,659 dogs of various breeds, although results were not reported separately for male and female dogs. Data validity may have been compromised due to owners reporting overweight/obese status on a questionnaire by comparing their dog’s body shape to five images, and because age was estimated in some cases for the shelter dogs. Confounding variables were reportedly controlled for, although it is unclear which variables specifically were included in the analyses. Simpson et al. [18] examined data for over 2,700 Golden Retrievers, however the exact number of dogs, and the number neutered in each age group was not clear. The results suggested no difference in age at neuter between those neutered at <6 and those neutered at 6–12 or >12 months of age. However, dogs neutered at 6–12 months had an increased risk for overweight/obesity compared to those neutered at >12 months of age. Dogs that were overweight prior to neutering were excluded, data were reported by veterinarians on an annual questionnaire and were based on a body condition scoring system, and confounding variables such as sex, physical activity level and age were controlled for.

Urogenital disease.

Six of the 18 studies that investigated urogenital disease (four UI, one USMI, one vulval development) considered pubertal status at the time of neutering [22, 24, 25, 46, 51, 56]. One of these [46] did not have a large enough sample size to enable statistical comparisons, and two reported no significant effects [24, 25]. The strengths and risk of bias for these two studies have previously been reported by Beauvais et al. [2]. Three studies did report effects of neutering before or after puberty on urogenital disease [22, 51, 56]. Stocklin-Gautschi et al. [56] suggested that the incidence of UI in bitches neutered prepubertally was lower than for bitches neutered post-pubertally, however there were issues with study methodology and a risk of bias that may compromise the results. The study only included bitches neutered before puberty and compared data to those for bitches that were neutered after puberty in a different study. The breeds and source of the bitches included and how outcomes were measured were not stated and the controlling for effects of confounding variables was not clear [2]. Lutz et al. [22] reported the opposite, with significantly more bitches neutered before puberty being reported with UI than those neutered after puberty. However, whether bitches with UI before neutering were excluded was not stated, UI was reported on an owner questionnaire and no OR or CI were reported. Importantly, results may be compromised by the grouping of bitches. There were some bitches aged up to 1.4 years in the group neutered before puberty and some aged as young as 0.3 years in the group neutered after puberty (presumably these are data errors).

The final study reported effects on vulval size and appearance for 306 Labrador / Golden Retriever crossbreed bitches [51]. Significantly more bitches neutered prepubertally had vulvas that changed to be smaller by 17 months of age, had smaller changes in vulval length and width between six and 17 months of age and had vulvas that appeared juvenile or recessed. This study considered vulval size and appearance prior to neutering and accounted for confounding variables in the analysis of vulval size.

The remaining 12 studies investigated the effects of timing of neutering related to age. Four of these made no comparisons of disease incidence between neuter age groups, only between each neuter age group and a group of entire dogs [15–17, 45]. Eight studies did make comparisons and three of those reported no impact of neutering age on urogenital disease [11, 26, 54].

Five studies reported effects of age at neutering on urogenital disease, specifically on recessed vulva [52]; cystitis and UI [12], and UI [47, 53, 58]. The only study to report more favourable results for bitches neutered earlier was a study examining UI onset post-neuter using data from veterinary records for 750 neutered bitches [58]. While the authors reported that the age at neuter and UI onset were related, with bitches neutered at older ages having earlier onset UI, they suggest that this “seems to confirm the lower risk in early spayed bitches compared to bitches spayed after the first oestrus, as reported by Stocklin-Gautschi et al. (2001)”. However, the statistical evidence to support this statement is weak, with supporting data not presented in the main section of the results and no description of confounding factors included in statistical analysis.

The four papers that reported less favourable findings for bitches neutered at younger ages examined UI, cystitis and recessed vulva. Spain et al. [12] reported significantly higher incidence of cystitis in bitches neutered before 5.5 months of age and increasing incidence of UI with decreasing age at neuter, with the highest risk of UI for bitches neutered before three months of age. UI was only considered when bitches required medical treatment. The number of bitches in each neuter group was not stated. Dogs were of various breeds and crossbreeds, however pure/crossbreed status were included in the analysis as cofounding variables. It is not clear how reliable age at neuter determination was for the dogs that were in the shelter at the time of neutering. Byron et al. [47] also investigated UI and found a decreased hazard for UI in bitches neutered at older ages that was significant for dogs weighing over 25 kg. This unmatched case-control study did not consider, or exclude bitches based on, health prior to neutering. Dogs were recruited via invitations to contribute data that were sent to 500 veterinary hospitals in the USA. The lack of matching of case-control dogs and the lack of controlling for confounding variables such as breed in the analysis affect the risk of bias in the study. Pegram et al. [53] also identified a relationship between age at neuter and early-onset UI when data for 23,499 neutered bitches were examined. In that study neutering before six months of age was associated with an increased hazard of early-onset UI. Data for a large number of bitches from UK primary care practices were examined with confounding variables including breed and bodyweight included in the analysis.

Finally, Palerme et al. [52] reported that recessed vulva, determined by direct observation of lateral and dorsal vulvar skin fold coverage, was significantly more likely in bitches neutered at or less than 12 months or 24 months of age, compared to those neutered over these ages. However, no differences were identified when age at neuter was grouped as less or greater than four or eight months of age. The study included various breeds and bitches were recruited from a university veterinary medical centre. Simple statistical analyses were performed with no controlling for confounding variables. The method of obtaining thorough history is unclear, therefore it is not obvious whether the approximate age at the time of ovariohysterectomy used for analysis was based on owner recollection or from veterinary records.

The effects on bitch health of the timing of neutering in relation to puberty have been investigated in only a small number of studies. Other studies have suggested that neutering before or after puberty has been examined, however on closer examination, age at neuter and not pubertal status has been used to categorise neuter timing [11] or the reported ages at neuter for the prepubertal and post-pubertally neutered groups suggest a risk of bias [22].

In summary there are few studies that investigate the health implications of neutering bitches considering pubertal status at neutering. Most studies that do consider pubertal status investigate urogenital health, commonly UI, and are subject to a risk of bias. Studies generally propose that neutering bitches earlier appears to be protective for some (but not all) cancers but increases the risk of DOD and potentially UI; contrasting results which make applying the findings to benefit bitch health difficult.

The limitations of this scoping review are that the database searches were conducted in May 2023. Since that time, further studies may have become available. Additionally, the search strategy may not have identified all of the literature, however the databases used are reported to provide high levels of coverage of veterinary literature [44]. This review was completed by two individuals, which was deemed appropriate for the volume of literature generated for review. The search terms and strategy were reviewed by a group of five specialists, one with extensive experience of conducting scoping reviews. Additionally, the inclusion of a librarian helped to optimise the search strategy [28, 40, 68, 69] and papers were included that were found in a previous literature search by the primary author and did not appear in the search results. Therefore, it is more likely that the search would have been too broad than failing to identify relevant articles. The search term ‘spey’ was not included which could have resulted in articles being missed if they did not also include other terns relating to neutering that were in the search terms.

A scoping review rather than systematic methodology was chosen due to the broad question posed and the need to examine and describe the potentially large range of literature that had not previously been synthesised [38, 40], at least for three of the five health outcomes included. The methodology was appropriate to provide an overview of the available and relevant literature and to identify gaps in the research [39, 41, 70, 71] and meant that the papers related to age at neuter could be included and summarised, rather than just the small number of papers that considered puberty, as would have been considered in a systematic review. This provided a broader overview of the available evidence and identified problems relating to features of the study methodologies. This allowed reporting of the factors relating to study design and characteristics, and recommendations to be made for future studies. The review also identified that there is insufficient literature for systematic reviews to be undertaken for most of these diseases.

Future studies that aim to examine the effects of neutering bitches before or after puberty should:

• be undertaken for a variety of dogs (pet, assistance, shelter etc.) from many geographical locations,
• consider breed and body weight, which can impact health outcomes,
• clearly define inclusion criteria including whether diseases are diagnosed before or after the neutering intervention, which should also be defined,
• randomly allocate dogs to neutering groups where all bitches are neutered using the same surgical method,
• consider study population size in order to ensure that the minimum number of bitches needed for valid statistical analyses are recruited, considering the likely disease incidence,
• use power analysis to determine appropriate values of alpha for the study population,
• identify and define puberty, considering the onset of puberty rather than age.

For studies examining the effects of timing of neutering relating to age, previous breeding history should be considered. Additionally, consideration of other studies when selecting age groups could enable easier comparisons to be made between studies.