PIOTR BARANOWSKI, MAGDALENA WRÓBLEWSKA, AND JOANNA WOJTAS

Bull Vet Inst Pulawy 53, 291-298, 2009 MORPHOLOGY AND MORPHOMETRY OF THE NUCHAL PLANE OF BREEDING CHINCHILLA (CHINCHILLA LANIGER, MOLINA 1782) SKULLS ALLOWING FOR SEX AND LITTER SIZE AT BIRTH PIOTR BARANOWSKI, MAGDALENA WRÓBLEWSKA, AND JOANNA WOJTAS Department of Animal Anatomy, Faculty of Biotechnology and Animal Husbandry, Agricultural University, 71-466 Szczecin, Poland Piotr.Baranowski@biot.ar.szczecin.pl Received for publication December 05, 2009 Abstract The aim of the investigation was to determine the effect of sex and litter size at birth on the values of selected metric traits of breeding chinchilla skulls. Eighty-six chinchillas of both sexes, aged 12 months, kept under similar nutrition and care conditions, were used in the study. The skull s weight was determined, as well as 14 craniometrical traits of the braincase and the splanchnocranium. The volume of the neurocranium and the area of the foramen magnum surface were determined, and the cranial index was estimated. The basic statistical measurements and the simple, parietal, and semi-parietal correlations between selected traits of the nuchal plane were estimated. The sex and litter size at birth were not the source for variations in the traits: A-P, B-P, A-N, N-P, Zyg- Zyg, Eu-Eu. A significant effect (P 0.01) of sex of the chinchilla on the height of the squama occipitael bone and the foramen magnum index was found. The inherence of the statistical significance in parietal and semi-parietal correlations for the selected nuchal plane of the skull confirms the necessity of making allowances for sex in craniometric research, as well as for litter size at birth in the case of estimating the values of the metric traits in the skulls of animals coming from multiple litters. Key words: chinchilla, skull, craniometry, morphometry, neurocranium, nuchal plane, foramen magnum. Opinions on the role of sexual dimorphism in animal size formation have been repeatedly raised in literature. A critical look at the skeleton of both sexes suggests that the grouping together of male and female skulls during the comparison of populations leads to the loss of a great deal of biological information (11). There are research results available in literature that confirm sexual dimorphism in cranial skeleton traits (3, 4, 7, 9, 10), but it is difficult to capture differences being statistically significant with respect to most metric traits even though male skulls, being larger, have stronger developed attachments for muscles and fasciae, with the attachment areas being larger as well. Another issue is the nuchal plane of the skull, including the shape of the foramen magnum in the contour of which one may observe, for instance in dogs, the presence of a dorsal notch or/and the presence of a bony bridge dividing this foramen. In some cases, this was considered to be a pathological change, especially as some changes in the neurological character were also stated during anamnesis (12). The dissimilarity of this foramen has not always induced pathological conditions, but undoubtedly is evidence of the inter-individual variability of a given population (15). Attempts should be made to try and find out the reasons for its appearance even in the prenatal period, as it most likely results from incomplete ossification (2). One cannot exclude, either, the fact that one of the reasons for the appearance of differences in the shape of the foramen magnum is the rate of skull growth (10), which may cause delays in the growth of the occipital bone and forces the development of small peripheral ossification centres. The presented paper shows the macroscopic analysis of the nuchal plane of breeding chinchilla skulls. Like in dogs (6), there was also observed in this species a variation in the contour of the foramen magnum as well as, in connection with that, its different width and height, and its effect on the size of the squama of the occipital bone. Bearing in mind the fact that the skull formation process and its ossification starts as early as embryogenesis and fully develops during postnatal life, with an important part played by sexual hormones, it was decided to examine whether a connection exists between sex and litter size at birth, and selected metric skull traits in the somatically developed breeding chinchillas. Material and Methods The study material consisted of 96 skulls of the long-tailed chinchilla (Chinchilla laniger, Molina 1782) aged 10-12 months, kept under similar nutrition and care

292 conditions on a breeding farm. Before decapitation, the carcass weight was determined by means of electronic scales exact to 0.01 g, as well as animal sex. The chinchilla heads were macerated by cooking them at about 100 o C for 1 h, followed by the removal of soft tissues muscles, nerves and blood vessels from the bone foramina. After rinsing them in running water, the skulls were dried at room temperature. In order to obtain onecolour white objects and expose all anatomical traits, the specimens were placed in 30% hydrogen peroxide solution for about 10 min. By means of electronic scales, the weight of each skull was determined, while, by using electronic slide callipers and craniometric points, the cranium dorsal part and base lengths and breadths (Akrokranion Prosthion; Akrokranion Nasion; Basion Prosthion; Nasion Prosthion; Zygion Zygion; Euryon Euryon) were measured exactly to 0.01 mm according to the method described previously (1), as well as those of the nuchal plane at the following points: 1. Basion- Bregma. 2. Height from Basion (B) to the external occipital protuberance of the occipital bone. 3. Height of the squama of the occipital bone. 4. Least breadth of the occipital bone. 5. Greatest breadth of the occipital condyles. 7. Greatest breadth of the paracondylar processes. 8. Breadth of the foramen magnum. 9. Height of the foramen magnum (Fig. 1). The obtained measurement results allowed us to derive the following cranial indices: Cranial index (neurocranium index)=eu-eu x 100 /A-N; Index 1=Eu-Eu x 100/A-P. Index 2=Eu-Eu x 100/B-P; index = Height F.m. x 100/Breadth F.m. Area of the occipital triangle according to an equation: P=a x h/2, where: a greatest breadth between paracondylar processes - 6; h height from Basion to the external occipital protuberance of the occipital bone -2. The volume of the neurocranium (cranial volume), which was determined by filling up the skull with selected rape seeds (with respect to their size and shape) through the foramen magnum, which was then measured in a measuring cylinder exact to 1 mm 3. The area of the foramen magnum surface ( area) was determined using MultiScan computer software, by means of which calculations were made. With this end in view, each skull was placed vertically, with the rostrum downwards, under a digital camera (Sony Alpha-100 with Tamron SP Di AF 90 mm 1:2.8 MACRO 1:1 lens) put on a calibrated frame. The position of the skull was exactly the same, so that its plane, created by the area of foramen magnum lumen, was perpendicular and horizontally positioned to the camera lens. The results of the metric measurements, skull volume and foramen magnum area, as well as the information concerning sex and litter size at birth, were entered into a Statistica v. 7 PL database. The mean values, standard deviations, and range were calculated for the value of each trait, as well as the coefficients of variation. The statistical analysis showed the normality of distribution for the examined traits, while differences between the male and female groups, allowing for litter size at birth, were examined using Tukey s test with an analysis of variance (ANOVA) for factorial designs. Between the selected metric traits and indices, the values of coefficients of simple, partial, and semi-partial correlations were calculated for P 0.05 and P 0.01. The collected bone material used in the study is housed in the museum collection of the Department of Animal Anatomy. Results The basic craniometric traits of the chinchilla skulls used in the study are presented in Table 1. Sex and litter size at birth were not the source of variation for the obtained values. The statistical analysis showed that interactions between sex and litter size at birth were nonsignificant; therefore this statistical relationship was ignored in the interpretation of the results. The analysis of the shape of the foramen magnum in 96 chinchilla skulls showed that 57 specimens of both sexes (59.37%) were characterised by a circular or round-like shape of the foramen magnum. In these skulls, no incisions were found in the foramen contour either (Fig. 2a). In the remaining 39 specimens (40.62%), the shape of the foramen magnum strongly differed from being circular and was characterised by incisions in the dorsal and lateral parts (Fig. 2b). The incision of the foramen magnum assumed a shape from small to large, entering high into the squama of the occipital bone. No osseous bridge, separating the foramen magnum, was found in any examined skull. The measurements of metric traits performed on the nuchal plane of the skull showed that females had a statistically significantly longer squama of the occipital (P 0.01) than males, and a consistently lower value of foramen magnum index (Table 2). The values of the remaining traits estimated for males and females did not differ statistically. The correlation between the index of the foramen magnum and its area (Fig. 3) was positive (r xy =0.62) and statistically significant (P 0.01). For the skulls of animals included in the group 1 due to foramen magnum shape, the index of this foramen amounted to 91.22% ± 6.65 and was by 13.39% smaller from the value estimated for the skulls of animals included in the group 2 (P 0.01). On the other hand, its value estimated for males exceeded by only 5.03% the value for females (P 0.01). A weak correlation was found between the value of the foramen magnum index and the estimated indices and the least breadth of the squama of the occipital bone, as well as a negative statistically significant (P 0.05) correlation with the area of the squama of the occipital bone and the cranium volume in males (Table 3). The correlation between the area of the foramen magnum surface and the height of the squama of the occipital bone assumed different values for both sexes, with positive ones for males (r xy =0.46) and negative ones for females (r xy = -0.38), but being statistically significant for both sexes (P 0.01). The coefficients of simple correlation for the indices and the breadth and height of the foramen magnum assumed low values, positive or negative, differently for females and males. For this reason, partial and semi-partial correlations were estimated in order to determine the strength of the relationship between the examined craniometric traits, allowing in the model for sex (Table 4) or litter size at birth (Table 5), as well as for sex and litter size at birth (Table 6).

293 Br 3 2 8 7 4 1 B 5 6 Fig. 1. Nuchal view. Craniometric measurements made on the skull from a Chinchilla laniger. Fig. 2. The caudal aspects of the skull from a Chinchilla laniger. Notice the wide variation in size and shape of the foramen magnum: 2a (DSC03467) - a circular or round-like shape of the foramen magnum; 2b (DSC 03488a) - the shape of the foramen magnum strongly differed from being circular and was characterised by incisions in the dorsal and lateral parts.

294 294 Table 1 Basic dimensions of the dorsal and basal part lengths and breadths (mm) of chinchilla skulls in males (n=49) and females (n=47) allowing for litter size at birth (x±s, min. max., V%) Trait Skull weight (g) A-P B-P A-N N-P Zyg-Zyg Eu-Eu Males Females singular litter (n=11) multiple litter (n=38) singular litter (n=13) multiple litter (n=34) 5.78 ± 0.40 (5.00 6.80); 6.96% 5.79 ± 0.58 (4.30 7.50); 10.06% 5.67 ± 0.47 5.82 ± 0.39 5.90 ± 0.53 5.80 ± 0.63 60.44 ± 1.60 (56.34 63.62); 2.65% 60.78 ± 1.85 (55.10 66.38); 3.06% 59.98 ± 2.22 60.58 ± 1.47 61.01 ± 1.22 60.66 ± 2.15 53.61 ± 1.32 (50.52 56.09); 2.47% 53.30 ± 1.70 (48.59 57.85); 3.19% 53.03 ±1.59 53.39 ± 1.35 53.22 ± 1.49 53.01 ± 2.05 41.61 ± 1.21 (38.15 44.08); 2.91% 41.88 ± 1.59 (37.43 46.82); 3.80% 41.31 ± 1.62 41.72 ± 1.11 41.70 ± 1.10 41.86 1.82 20.65 ± 1.10 (17.93 22.83); 5.35% 20.82 ± 1.02 (18.42 23.66); 4.34% 20.16 ± 1.63 20.76 ± 0.96 20.95 ± 0.83 20.77 ± 1.00 32.20 ± 0.74 (30.74 33.55); 2.31% 32.37 ± 1.12 (30.16 37.23);3.45% 32.30 ± 0.87 32.16 ± 0.74 32.74 ± 0.69 32.33 ± 1.28 25.24 ± 1.01 (23.17 29.92); 3.98% 25.46 ± 0.81 (24.04 27.36); 3.19% 25.22 ± 0.60 25.26 ± 1.11 25.52 ± 0.78 25.47 ± 0.92 Table 2 The values of selected metric traits (mm), indices (%) and surface areas (mm 2 ) for the nuchal plane of chinchilla skulls (x± s;min. max.) Trait Males (n=49) Females (n=47) Height of skull from B to Br 20.17 ± 0.73 (18.09 21.65) 20.15 ± 0.77 (18.64 22.27) Height from B to external occipital protuberance 14.17 ± 0.96 (11.74 15.78) 14.35 ± 0.64 (13.10 15.68) Height of occipital squama 6.29** ± 0.95 (4.37 8.19) 6.78** ± 0.81 (5.35 9.01) Least width of occipital bone 10.67 ± 0.93 (8.93 13.39) 10.73 ± 0.71 (9.36 12.13) Breadth of occipital condyles 8.88 ± 0.45 (8.07 10.02) 8.97 ± 0.58 (7.78 10.35) Breadth between paracondylar processes 19.18 ± 0.88 (16.92 20.86) 19.14 ± 1.52 (13.10 21.37) Breadth of foramen magnum 7.71 ± 0.42 (6.79 8.61) 7.82 ± 0.43 (6.99 8.68) Height of foramen magnum 7.64 ± 1.09 (5.61 10.92) 7.36 ± 0.75 (5.78 8.86) index 99.12 ** ± 12.38 (77.00 131.00) 94.09 ** ± 7.88 (81.30 110.76) Cranial volume (cm 3 ) 5.15 ± 0.30 (4.50 6.00) 5.14 ± 0.35 (4.00 6.00) area 42.28 ± 5.96 (32.63 58.19) 41.95 ± 4.91 (31.87 52.42) Area of occipital triangle 135.97 ± 11.39 (105.32 162.29) 137.51 ± 14.06 (85.80 161.71) ** - P 0.01

295 Measurement Table 3 The value of selected coefficients of correlation for craniometric traits in chinchilla males and females area width height index m. f. m. f. m. f. m. f. Cranial index -0.13-0.09-0.30 * 0.11-0.19-0.13-0.09-0.24 Index 1 0.12 0.11-0.07 0.26 0.01 0.02 0.04-0.17 Index 2 0.11 0.06-0.09 0.22 0.04 0.07 0.08-0.08 Least width of occipital bone -0.04 0.35 * 0.28 * 0.27-0.12 0.17-0.26 0.05 Area of occipital triangle -0.30 * 0.04-0.23-0.14-0.29 * -0.03-0.22 0.06 Cranial volume -0.39 ** 0.17 0.49 ** 0.11 0.19 0.05 0.01-0.01 Height of squama occipitale 0.46 ** -0.38 ** -0.07-0.26-0.53 ** -0.57 ** -0.57 ** -0.53 ** m. males; f. - females * - P 0.05; ** - P 0.01 Table 4 Partial and semi-partial correlations for selected traits of the nuchal plane of chinchilla skulls allowing for sex-related variation Measurement area width height index rp rs-p rp rs-p rp rs-p rp rs-p Cranial index -0.333** -0.282** -0.261* -0.227* -0.308-0.237-0.202-0.156 Index 1 0.265* 0.219* 0.138 0.116 0.131 0.097 0.063 0.047 Index 2-0.015-0.012 0.046 0.039 0.100 0.073 0.090 0.068 Least width of occipital bone 0.022 0.017 0.244* 0.211* -0.121-0.089-0.261* -0.202* Area of occipital triangle -0.042-0.034-0.198-0.169-0.051-0.037 0.063 0.047 Cranial volume 0.359** 0.306** 0.288** 0.252** 0.327** 0.253** 0.211* 0.162* Height of squama occipitale -0.412** -0.360** -0.186-0.158-0.581** -0.522** -0.567** -0.518** Explanations: values of coefficients of correlation marked with * - significant at P 0.05; with ** - significant at P 0.01 295

296 296 Table 5 Partial and semi-partial correlations for selected traits of the nuchal plane of chinchilla skulls allowing for litter size at birth-related variation Measurement area width height index rp rs-p rp rs-p rp rs-p rp rs-p Cranial index -0.343** -0.291** -0.277** -0.246** -0.310** -0.236** -0.190-0.144 Index 1 0.250** 0.206** 0.098 0.084 0.137 0.100 0.089 0.067 Index 2 0.018 0.014 0.114 0.098 0.104 0.075 0.055 0.041 Least width of occipital bone 0.015 0.012 0.242* 0.212* -0.135-0.098-0.278** -0.216** Area of occipital triangle -0.032-0.026-0.199-0.173-0.030-0.022 0.091 0.068 Cranial volume 0.358** 0.306** 0.270* 0.239* 0.340** 0.261** 0.233* 0.179* Height of squama occipitale -0.409** -0.357** -0.019-0.016-0.610** -0.556** -0.613** -0.579** Explanations: values of coefficients of correlation marked with * significant at P 0.05; with ** - significant at P 0.01 Table 6 Partial and semi-partial correlations for selected traits of the nuchal plane of chinchilla skulls allowing for sex-related and litter size at birth-related variation Measurement area width height index rp rs-p rp rs-p rp rs-p rp rs-p Cranial index -0.405** -0.339** -0.324** -0.278** -0.338** -0.260** -0.199-0.153 Index 1 0.368** 0.302** 0.217 0.180 0.204 0.151 0.101 0.076 Index 2-0.083-0.063-0.003-0.002 0.047 0.034 0.058 0.049 Least width of occipital bone -0.028-0.021 0.218 0.181-0.160-0.117-0.276* -0.216* Area of occipital triangle -0.085-0.065-0.251* -0.210* -0.074-0.054 0.063 0.047 Cranial volume 0.376** 0.310** 0.294** 0.249** 0.325** 0.249** 0.203 0.156 Height of squama occipitale -0.391** -0.325** -0.168-0.139-0.555** -0.483** -0.535** -0.477** Explanations: values of coefficients of correlation marked with * - significant at P 0.05; with ** - significant at P 0.01

297 Fig. 3. The correlation between the index of foramen magnum and foramen magnum area. Discussion The analysis of the obtained values for the basic cranial indices did not show any effect of chinchilla sex and litter size at birth on the values of the basic metric traits that characterise their neuro- and viscerocranium, even though there are reports of reversed sexual dimorphism in this species. The breeding chinchilla females are heavier than males by about 200 g (13). The carcass weight of the animals randomly selected for examination did not differ statistically significantly, like the weight of skulls of both sexes. Differences that result from the effect of sex were repeatedly reported in literature (5, 8, 14), frequently admitting that sex determination, due to considerable morphological similarity, can be of great taxonomic importance. The results presented in Table 1 did not show any statistically significant differences in craniometric traits either, when the effect of litter size at birth was taken into consideration. On the other hand, the analysis of data from Table 2 points to a statistically significant (P 0.01) effect of sex on the height of the squama of the occipital bone but no effect of the litter size at birth was found in the value of estimated traits. The analysis of the size and shape of the foramen magnum in Pekingese dogs also showed no possibility for its use in the identification of skulls with regard to sex (12). The calculated index of the foramen magnum, used as a biometric criterion, aims at its proper morphological evaluation, and at the same time neutralises the effect of skull size. It also allows the comparison of the obtained results between animals of different size within a species. Because in the examined population no statistically significant differences were found between animals of both sexes, it was therefore decided to determine the strength of the relationship between selected traits that characterise the skull by means of partial and semipartial correlation matrices. It was found that, with respect to some of the examined traits, there is a statistically significant (P 0.01; P 0.05) dependency in the relationship of examined traits on sex, litter size at birth, and/or these both variables simultaneously (Tables 4-6). The study results obtained confirm the necessity of making allowance for sex in craniometric research, as well as for litter size at birth in the case of estimating the values of metric traits in the skulls of animals coming from multiple litters. References 1. Baranowski P., Wojtas J., Cis G., Musiał M., Wróblewska M., Sulik M.: Value of craniometrical traits in chinchillas (Chinchilla laniger) skulls considering teeth defects. Bull Vet Inst Pulawy 2008, 52, 271-280. 2. Berry A.C., Berry R.J.: Epigenetic variation in the human cranium. J Anat 1967, 101, 361-379. 3. Gittleman J.L.: Sexual dimorphism in the canines and skulls of carnivores: effects of size, phylogeny, and behavioural ecology. J Zool (London) 1997, 242, 97-117. 4. Günay Y., Altinkök M.: The value of the size of foramen magnum in sex determination. J Clin Forensic Med 2000, 7, 147-149.

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