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22001155 NORTHEASTERN NATURALIST 2V2(o4l). :2625,2 N–6o5. 74
Evaluation of a Formula that Categorizes Female Gray Wolf
Breeding Status by Nipple Size
Shannon M. Barber-Meyer1, 2,* and L. David Mech3
Abstract - The proportion by age class of wild Canis lupus (Gray Wolf) females that
reproduce in any given year remains unclear; thus, we evaluated the applicability to our
long-term (1972–2013) data set of the Mech et al. (1993) formula that categorizes female
Gray Wolf breeding status by nipple size and time of year. We used the formula to classify
Gray Wolves from 68 capture events into 4 categories (yearling, adult non-breeder, former
breeder, current breeder). To address issues with small sample size and variance, we created
an ambiguity index to allow some Gray Wolves to be classed into 2 categories. We classified
20 nipple measurements ambiguously: 16 current or former breeder, 3 former or adult
non-breeder, and 1 yearling or adult non-breeder. The formula unambiguously classified 48
(71%) of the nipple measurements; based on supplemental field evidence, at least 5 (10%)
of these were incorrect. When used in conjunction with an ambiguity index we developed
and with corrections made for classifications involving very large nipples, and supplemented
with available field evidence, the Mech et al. (1993) formula provided reasonably
reliable classification of breeding status in wild female Gray Wolves.
Introduction
Some important areas regarding reproduction in wild female Canis lupus L.
(Gray Wolf, herefter Wolf) are not yet fully understood. Although some female
Wolves breed at 1-y old (Medjo and Mech 1976, Seal et al. 1979, Sidorovich and
Rotenko 2014), some wild Wolves do not breed until they are 4–5 years old (Mech
and Seal 1987, Mech et al. 1998). The proportion of females in a population that
breed in any given year is unknown, but this metric is important because it is fundamental
to understanding Wolf population dynamics, is useful for modeling Wolf
populations (Miller and Lacey 2005), and it is required for calculation of generation
time in Wolf genetic research.
We were interested in whether an equation from an earlier study (Mech et al.
1993) designed to categorize female Wolf breeding status based on nipple sizes of
captive Wolves, could be applied to our long-term data set and enable us to determine
the proportion of female breeders in any year. Mech et al. (1993) recorded
nipple measurements of 29 captive Wolves with known breeding histories every 2
weeks and categorized them into 4 breeding classes (yearling, adult non-breeder,
former breeder, and current breeder). The mean and standard deviation of nipple
sizes were calculated for each category every 2 weeks with sample sizes ranging
1US Geological Survey, Northern Prairie Wildlife Research Center, 8711 37th Street SE,
Jamestown, ND 58401-7317. 2Current address - US Geological Survey, 1393 Highway 169,
Ely, MN 55731. 3Current address - The Raptor Center, 1920 Fitch Avenue, University of
Minnesota, St. Paul, MN 55108. *Corresponding author - sbarber-meyer@usgs.gov.
Manuscript Editor: John Litvaitis
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from 4 to 17 for each category each period. The researchers then developed the following
classification formula based on nipple size and time of y ear:
ts = (Y1 – Y2)/(s2 √[n2 +1]/n2),
Where, Y1 = length + width nipple measurement from Wolf, Y2 = sample mean from
appropriate period, s2 = sample deviation from period, n2 = sample size, ts = probability
of nipple measurement under Ho: same population, and n2 – 1 = degrees of
freedom. We applied this formula to wild Wolves captured in the Superior National
Forest in Minnesota, to determine the formula’s reliability and applicability. Mech
(2006) conducted a preliminary study of Wolf breeding status for various ages, but
that investigation involved only 23 Wolves distributed over ages 2–12 y and did not
directly apply the Mech et al. (1993) classifying formula (hereafter, the formula) as
we did in this study.
Methods
Our study area covered 2060-km2 in the Superior National Forest, MN, (48°N,
92°W; see Heinselman 1993 and Nelson and Mech 1981). As part of a long-term
study of Wolves and Odocoileus virginianus Zimmermann (White-tailed Deer;
Mech 2009), we captured wild Wolves with modified foot-hold traps (either Newhouse
14 or EZ Grip 7, Rancher’s Supply, Alpine TX) from 1972 to 2013 (Mech
2009) and followed guidelines of the American Society of Mammalogists (Gannon
and Sikes 2007) during capture and processing (see Barber-Meyer and Mech 2014
for details). We anesthetised all adult trapped Wolves (all non-pups) with a standard
dose of 250 mg ketamine (Ketaset1, ketamine hydrochloride; Fort Dodge Animal
Health, Fort Dodge, IA; 1988–1991) or 286mg telazol (R) (tiletamine hydrochloride
and zolazepam hydrochloride; Pfizer, New York, NY, and Fort Dodge Animal
Health; 1992–2011) and 37 mg xylazine (Anased(R); Llyod Laboratories, Shenandoah,
IA) given intramuscularly via a pole syringe (approximate length 4 feet [1.2
m]). An additional 100–200 mg of ketamine was handinjected intramuscularly if
required. We recorded standard morphological measurements, collected specimens
such as blood (0–28 cubic centimeter [cc]) and sometimes scat and hair, and applied
ear tags (jumbo rototag, Nasco, Fort Atkinson, WI; or sheep/goat tag, Destron Fearing,
Farmington, MN) and a VHF radiocollar (Telonics, Inc., Mesa, AZ). Beginning
in 2000, we used tooth wear to estimate Wolf age by comparing our samples with
the chart in Gipson et al. (2000). Prior to 2000, unless the Wolf was of known age
(i.e., captured as a pup), we assigned a known-minimum age of 1 year and updated
the age if a Wolf was recaptured. We administered antibiotics and an antagonist to
the anesthetic. We handled Wolves for ~1 h in total.
We excluded yearlings and pups from our analyses because it is not common for
Wolves less than 2 y old to breed. We classified Wolves with inconspicuous nipples as adult
non-breeders. We entered the observed nipple size for all measured Wolves into the
formula and added values corresponding to the appropriate period. We calculated
t-values for each of the 4 potential breeder categories and then used these values in
Arc version 1.06 (Cook and Weisberg 1999) to generate a 2-tailed probability (as
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detailed in Mech et al. 1993) that the observed nipple size belonged to the same
statistical population as each Mech et al. (1993) category. We classified the Wolf
in the category with the highest probability among all categories. We encountered
some problems when the second highest probability was very close to that of the
assigned category. Therefore, we created an ambiguity index for each Wolf by summing
all the resulting probabilities and determining what fraction of the total was
contributed by each category (contribution fractions summed to 1). If the difference
between the contribution fraction from the selected category and the next highest
probability category was less than an arbitrarily chosen value of 0.20, we categorized
the Wolf as potentially in either category (e.g., current or former breeder,
former or adult non-breeder, and yearling or adult non-breeder).
Results
We classified 86 individual Wolves 2 y and older into breeding categories based
on their nipple sizes. Sixty-eight capture events included Wolves with conspicuous
nipples that we measured and classified according to the formula. Of these 68,
we considered 20 (29%) to be ambiguous classifications (1 was adult non-breeder
or yearling, 3 were former breeders or adult non-breeders, and 16 were current or
former breeders). For example, one ambiguous classification was a 3-y-old, Wolf
(#7057) with a nipple size of 1.0 cm measured on 25 August 2008. For this period,
the Mech et al. (1993) mean nipple size for adult non-breeders was 0.85 cm (SD =
0.16, n = 17) and mean nipple size for former breeders was 1.45 cm (SD = 0.33,
n = 6). When we applied the formula and ambiguity index, we classified Wolf #7057
ambiguously as an adult non-breeder (unscaled probability of belonging to that category
= 0.375) or former breeder (unscaled probability of belonging to that category
= 0.262) because the difference between these top 2 categories in scaled contributing
probabilities was 0.145 (less than the 0.20 cutoff). We excluded known yearlings
from our analysis; thus, we concluded Wolf #7057 was an adult non-breeder. Three
Wolves classified as former or adult non-breeders. In 1 case, we had information
from a previous capture to conclude that the Wolf was actually a former breeder. Of
the 16 animals that were classified as current or former breeders using the formula
and ambiguity index, there were 2 cases in which current breeders were erroneously
classed ambiguously because they had large nipples, 1 case where pups were
observed (and the female was estimated at 4-y old), and 1 case where we concluded
that the 3-y old female was likely a current breeder because radio telemetry and visual
observations indicated that she was alone with her mate the previous winter, she
showed restricted movements based on her 3 locations during the beginning of the
denning period (suggesting she denned) before her radio-signal ceased, and because
her pack totaled at least 5 Wolves the following November (suggesting successful
reproduction).
Of the 68 classifications based on conspicuous nipple measurements, 48 (71%)
were unambiguous. Of the unambiguous classifications, at least 5 (10%) were
likely erroneous. Three of these were due to sample-size and rank-order issues.
Specifically, at 5-y, nipple size of Wolf #979 was 2.3 cm on 28 August 28 2005.
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For this period, the Mech et al. (1993) mean nipple size for current breeders was
1.52 cm (SD = 0.34, n = 14) and mean nipple size for former breeders was 1.45 cm
(SD = 0.53, n = 4). A larger sample and smaller SD of current breeders (relatively
narrower confidence interval) for this date resulted in Wolf #979 being categorized
erroneously as a former breeder (relatively wider confidence interval). Similarly, 2
other Wolves that were current breeders with larger nipple sizes were erroneously
classed as former breeders.
The remaining 2 incorrect unambiguous classifications included Wolves with
relatively smaller nipples that were classed as former breeders but were lactating.
For categorization, we considered lactating Wolves to be current breeders, but both
of these Wolves could have been pseudopregnant (Jochle 1977, Seal et al. 1979; but
see also Mech and Seal 1987).
We did not detect any other inconsistencies in the rest of the classifications as
compared to available supplemental field evidence (e.g., observed pups, female lactating,
female localizing during denning and rendezvous seasons, female the only
known adult female in the pack, etc.).
Discussion
In general, we determined that in wild Wolves, the formula reliably separated
yearlings and adult non-breeders from current breeders. It did not perform well at
separating current and former breeders, and also, to a lesser extent, former from
adult non-breeders. These problems were due to several factors: small samples in
the captive wolves used for comparison, the formula’s inability to account for the
order of nipple-sizes among categories (i.e., nipple sizes should generally be yearling
< adult non-breeder < former breeder < current breeder; thus, Wolves with very
large nipples should be placed in the current breeder category), inadequate information
on nipple regression, and individual variation in Wolves.
To increase the applicability of the formula, additional information is needed
on how quickly a former breeder’s nipple measurements return to the size-range of
an adult non-breeder. Four Wolves (ages 6, 7, 9, and 9) had inconspicuous nipples
(L.D. Mech and S.M. Barber-Meyer, unpubl. data). This finding raises the questions:
(1) were they incorrectly aged? (2) had they never bred? or (3) had they bred
and their nipples regressed? Future research may help to answer these questions.
We know that at least 1 of our Wolves had very small (0.6 cm) nipples at 7 y, but
bred at least when she was 2-, 3-, and 5-y old (L.D. Mech and S.M. Barber-Meyer,
unpubl. data). Results of studies of the mammary glands of domestic dogs would
likely help us understand nipple size in wolves; however, such studies have not
been conducted (Evans and de Lahunta 2013:399). Also, it would be useful to
gather additional data from Wolves from other regions where body size differs from
the Wolves we evaluated for this study to determine if the formula applies equally.
Some uncertainty in classifying Wolves will remain regardless of refinements
to the formula and increases in the sample of known-breeding status wolf nipple
sizes simply due to inherent variation among individual Wolves. As a supplement
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to the classifications generated by the formula, researchers may wish to use available
field evidence to class some females as current breeders if they are known to
be the only adult female in their pack and have been observed with pups or had
localizing movements during denning and rendezvous season, etc. Additionally, we
recommend that during necropsies, researchers collect placental-scar information
whenever possible along with nipple sizes for comparisons.
Acknowledgments
This project was supported by the US Geological Survey. We thank M.E. Nelson and
numerous volunteer technicians. We are grateful to the sponsors and organizers of the
European Large Carnivores: Problems of Small-sized Populations, Study on Reproduction,
and Challenges of Reintroduction Programs Conference in Krasny Bor, Belarus, September
2014. We thank Lori Schmidt (International Wolf Center and Vermilion Community College,
Ely, MN) and Ellen Heilhecker (Washington Department of Fish and Wildlife) for
reviewing an earlier draft. Any mention of trade, product, or firm names is for descriptive
purposes only and does not imply endorsement by the US government.
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