Pesq. Vet. Bras. 34(1):83-90, janeiro 2014

Morphological and morphometric evaluation of prepubertal gilt , uterine tubes and at different oestrus cycle stages1

Guilherme Oberlender2, Thais P. Pontelo3, José R. Miranda4, David R. Miranda4, Márcio G. Zangeronimo3*, Adriana C. Silva3, Tila A. Menezes3 and Luiz G.P. Rocha3

ABSTRACT.- Oberlender G., Pontelo T.P., Miranda J.R., Miranda D.R., Zangeronimo M.G., Silva A.C., Menezes T.A. & Rocha L.G.P. 2014. Morphological and morphometric eval- uation of prepubertal gilt ovaries, uterine tubes and uterus at different oestrus cycle stages. Pesquisa Veterinária Brasileira 34(1):83-90. Setor de Fisiologia e Farmaco- logia Veterinárias, Departamento de Medicina Veterinária, Universidade Federal de La- vras, Campus Universitário s/n, Caixa Postal 3037, Lavras, MG 37200-000, Brazil. E-mail: [email protected] Studies are performed in developing techniques/procedures that provide greater repro- ductive performance in farm animals, including pigs. In this sense, the study of gilts repro- ductive organs at different oestrus cycle stages for assessing the presence of abnormalities and/or other parameters that may affect the future animal fertility is important. In order to evaluate the morphological, morphometric and histomorphometric features of ovaries, uterus and uterine tubes (UTs) characteristics of prepubertal gilts at different oestrus cycle stages, reproductive tracts from 48 animals immediately after slaughter were obtained. Af-

of oestrus cycle stage of each gilt in follicular phase (FP) and luteal phase (FL). Then, mor- phometricter, the structures evaluations were dissectedof ovaries, and UTs, removed, uterine andhorns the and ovaries uterine were body used were for classification performed.

- nedBesides and that,stained medial with segments Hematoxylin of UTs and and Eosin. uterus Histomorphometric were fixed in Bouin analyzes solution, using processed image and included in paraffin, when histological sections of 5.0 micrometers (µm) were obtai Student’s oestruscapture cyclesystem stages and specific(FP × LP) software and between were performed.the placement Afterwards, of reproductive data were structures submitted (right to t test for assessment the statistical differences (P<0.05) between the two different

in× leftFP andantimer). LP. Likewise, Among inthe respect gilts evaluated, to the placement 35 were of in reproductive the FP and 13 structures, in LP. There both was in theno difference (P>0.05) between morphometric parameters of ovaries, UTs and uterus of gilts

ofoestrus glandular cycle and stages, UT epithelium as in the general compared average, to animals there was in LP. no On difference the other (P>0.05). hand, the Regarding diameter the histomorphometric variables, gilts classified in FP presented a higher (P<0.05) height

of endometrial glands was higher (P<0.05) in gilts at LP compared to FP. Furthermore, gilts conclusion,in LP presented in prepubertal a higher (P<0.05) gilts, the proportion histomorphometric of parameters occupied as endometrial by glands, whereas glands diameter,animals in the FP heighthad a higherof glandular (P<0.05) epithelium proportion and of of connective UT epithelium tissue and and the blood proportion vessels. Inof

1 Received on February 7, 2013. 3 Setor de Fisiologia e Farmacologia Veterinárias, Departamento de Me- Accepted for publication on January 9, 2014. dicina Veterinária, Universidade Federal de Lavras (UFLA), Campus Uni- 2 Curso de Medicina Veterinária, Universidade Federal da Fronteira Sul versitário s/n, Cx. Postal 3037, Lavras, MG 37200-000, Brazil. *Correspon- (UFFS), Campus Realeza. Acesso Rodovia PR-182 Km 466, Rua Edmun- ding author: 4 Setor de Morfologia Veterinária, Departamento de Medicina Veterinária, [email protected] UFLA, [email protected] Universitário s/n, Cx. Postal 3037, Lavras, MG 37200-000. do Gaievski, 1000, Bairro Jardim Primavera, Cx. Postal 253, Realeza, PR 85770-000, Brazil. E-mail: 83 84 Guilherme Oberlender et al.

endometrium occupied by connective tissue, besides the glands and blood vessels varies

INDEX TERMS: Follicular phase, histology, luteal phase, morphology, morphometry, reproduction, reproductivethrough the tract,oestrus swine cycle,. possibly under the influence of ovarian steroids.

RESUMO.- [Avaliação morfológica e morfométrica de INTRODUCTION ovários, tubas uterinas e útero de fêmeas suínas pré-pú- In animal production, when studying the female reproduc- beres em diferentes fases do ciclo estral.] Pesquisas são tive tract, one of the most important aspects is the herd re- realizadas no desenvolvimento de técnicas/procedimentos productive performance, and this is a technical approach - produção, dentre esses, os suínos. Nesse sentido, o estudo ming (Monteiro & Carvalho 2006). This fact is important, que propiciem maior eficiência reprodutiva em animais de oncewith profitfertility purpose, disorders which may is lead the mainto the objective gilts culling of pig resul far- fases do ciclo estral é importante para avaliação da presen- ting in lose of productivity (Heinonen et al. 1998). çados de órgãos anomalias reprodutivos e/ou demais das fêmeasparâmetros suínas que em possam diferentes afe- - tar a fertilidade futura do animal. Objetivando-se avaliar as ad technique used to reduce production costs and to achie- características morfológicas, morfométricas e histomorfo- ve aIn better pig farming, use of thethe artificial male gametes insemination into porcine is a widespre (Severo - et al. 2011). Furthermore, in order to achieve high produc- as suínas pré-púberes em diferentes fases do ciclo estral, tivity indices (weaned piglets), the gilts should be prepa- tratosmétricas reprodutivos dos ovários, foram útero obtidose tubas uterinasde 48 animais (TUs) de imedia fême- tamente após o abate. Posteriormente, as estruturas foram the and parturition installations, and also being dissecadas e retiradas, sendo os ovários utilizados para a red for insemination and thus, making an efficient use of 2012). However, this situation does not always happens in folicular (FF) e fase luteínica (FL). Em seguida, foram rea- practice.prepared to replace the slaughtered sows (Stančić et al. lizadasclassificação avaliações da fase morfométricas do ciclo estral dos de ovários,cada fêmea TUs, em cornos fase Approximately 30 to 40 % of gilts does not show signs uterinos e corpo uterino. Após, segmentos mediais das TUs of oestrus, even after 8 months of age, and, these animals - - nation as gilts that present delayed puberty, namely, gilts in e útero foram fixados em solução de Bouin, processados e are slaughtered, on their majority, before the first insemi comincluídos Hematoxilina rotineiramente e Eosina. em parafina,Análises quandohistomorfométricas cortes histo foramlógicos realizadas de 5,0 micrômetros utilizando sistema(µm) foram de captura obtidos de e imagenscorados femalesprolonged removal anestrus is related pre-insemination to reproductive (Stančić failure et al. (López 2008,- - -SerranoGagrčin et et al. al. 2009). 2000, InDiehl addition, et al. 2003). among Given 13 to this, 49 % one of swineof the ram submetidos ao teste t de Student para a avaliação de current challenges in pig production systems is maintain e software específico. Posteriormente, os dados obtidos fo constant the level of gilts productivity. ciclo estral (FF × FL) e entre a localização das estruturas re- Some of the main components of the female reproductive diferenças estatísticas (P<0,05) entre as diferentes fases do tract are uterus and uterine tubes (UTs), essential organs for reproduction, and its growth is regulated by several factors produtivas (antímero direito × esquerdo). Dentre as fêmeas avaliadas, 35 encontravam-se na FF e 13 na FL. Não houve- number of variations according to the animal’s age and to the ma,diferença em relação (P>0,05) à localização entre os parâmetros das estruturas morfométricos reprodutivas, dos occurrence(Spencer et ofal. 2005). These (Monteiro structures et al.undergo 2003, Guimarãesby a large tantoovários, nas TUs duas e útero fases das do fêmeasciclo estral, nas FF como e FL. na Da média mesma geral, for et al. 2004). In pigs, studies have been performed analyzing - the behavior of uterus and endometrium related to the fer- - tility (Johnson et al. 2009, Bazer et al. 2012). Regarding the não houve diferença (P>0,05). Em relação às variáveis his UTs, few studies have been done (Guimarães et al. 2004, comparaçãotomorfométricas, com asos fêmeasanimais classificadas na FL. Por outro na FF lado, apresenta o diâ- Tienthai et al. 2006), although it is essential at transport and ram maior (P<0,05) altura do epitélio glandular e da TU em interaction between gametes, at maintenance of sperm and oocytes viable until the fertilization and spermatic capacita- metro das glândulas endometriais foi maior (P<0,05) nas- tion (Rodriguez-Martinez et al. 2001, Coy et al. 2008). porçãofêmeas classificadasdo endométrio na FLocupada em comparação por glândulas, com aenquanto FF. Além Besides the aforementioned organs, the ovaries also are disso, as fêmeas na FL apresentaram maior (P<0,05) pro- part of the female reproductive tract. These are in pairs, ção de tecido conjuntivo e vasos sanguíneos. Em conclusão, with varied placement and size between animal species que as fêmeas da FF apresentaram maior (P<0,05) propor- and are responsible for the production of gametes and hor- métricos, o diâmetro das glândulas endometriais, a altura mones (Hafez & Hafez 2004, Câmara & Cunha Dias 2008). doem epitélio fêmeas glandularsuínas pré-púberes, e da TU e a os proporção parâmetros do histomorfoendométrio The assessment of morphological characteristics of the ocupada por tecido conjuntivo, além das glândulas e vasos ovaries may be used to investigate the presence of patho- sanguíneos variam ao longo do ciclo estral, possivelmente logies such as cysts and tumors, and may also be useful in order to estimate which is the probable oestrus cycle stage TERMOS DE INDEXAÇÃO: Aparelho reprodutor, fase folicular, fase and, this way, can affect the future fertility of the animal luteínica,devido à histologia,influência morfologia, dos esteróides morfometria, ovarianos. reprodução, suíno. (Nascimento et al. 2003, Ramos et al. 2008).

Pesq. Vet. Bras. 34(1):83-90, janeiro 2014 Morphological and morphometric evaluation of prepubertal gilt ovaries, uterine tubes and uterus at different oestrus cycle stages

85 Studies of the morphological, morphometrics and histo- Morphological and morphometric evaluation morphometrics characteristics of the reproductive organs For morphological and morphometric evaluation of the ova- of nulliparous females in different oestrus cycle stages are important as a tool for assessing the reproductive status of (LO). After, they were measured at larger (major axis) and lowerries, it lengthwere removed (axis between and identified ovarian in pedicle right ovary and (RO)the opposite and left extremity) using a caliper (graduated in millimeters - mm), res- considering that gilts are the replacement animals and that pecting the organ symmetry (Nascimento et al. 2003, Câmara & theythese often animals present before reproductive the first insemination disorders, which and pregnancy, increases Cunha Dias 2008, Ramos et al. 2008). For the assessment of the the production costs. weight (grams), in each gilt the RO and LO was individually wei- Considering the above, this study aimed to evaluate the ghted using electronic digital analytical balance (capacity = 220g, morphological, morphometrics and histomorphometrics accuracy of 0.01g, AY-220, Shimadzu, Japan). characteristics of the ovaries, uterus and UTs of prepuber- The UTs and uterine horns were dissected of the tal gilts in different oestrus cycle stages. and , isolated and the length of the right and left antimer of each structure measured (Tummaruk et al. 2009). For MATERIALS AND METHODS UTs, the length was estimated from the UT abdominal ostium to the uterine ostium of the UT. For uterine horns, the measurement Ethics was initiated of the light of each horn, from the uterine body, to The present study was conducted after the institutional ap- the beginning of each UT (Santos et al. 2000). As to the ovary, the proval of the Federal University of Lavras (UFLA), with all expe- UTs and uterine horns were individually weighted. rimental protocols being compliant with the Ethical Principles of Animal Experiments adopted by the Institutional Animal Care and Histological preparation Use Committee Guidelines of this institution, under the protocol number 009/10. morphological and morphometric evaluations, the histological preparationAfterwards of biologicalthe classification materials of theobtained oestrus was cycle performed. stage and The the Place of investigation and location of animals slides preparation was performed at the laboratory of Veterinary The study was performed at the laboratories of Veterinary Morphology (DVM/UFLA) from the samples of horns and UTs of Physiology and Pharmacology and Veterinary Morphology, lo- each animal previously obtained. cated at the Department of Veterinary Medicine (DVM) at UFLA. For histological evaluation, fragments of approximately 2.0cm All biological material was obtained from a slaughterhouse in length of the medial portion of both uterine horns, right and ( ) situated in Lavras city, Mi- - nas Gerais state, Brazil, during the months of May to October pulla UTs region were collected. Following, the material obtained 2012.“Matadouro/Frigorífico A total of 48 Landrace NUTRILI” × Large White prepubertal gilts left, and segments of approximately 0.5cm of the right and left am to 6 months and weighing between 90 to 100 kg on average were hourswas fixed (Eigenheer-Moreira in Bouin solution et (75 al. %2007, saturated Moraes picric et al. acid 2007, solution, Aleixo used.(lineage cross-bred commercial - Genetics AGROCERES) aged 5 et25% al. formaldehyde2011). Then, the and samples 5% glacial were acetic dehydrated acid) for with a period increasing of 24 concentrations of ethanol, cleared in xylene and included in pa- Oestrus cycle classification - For each slaughtered female, the reproductive tract was col- 2006)raffin (Drummond were obtained, et al.mounted 2004). Afterwards,on microscope transverse slides and cross-sec stained phase (FP) or luteal phase (LP), according to the methodology withtions Hematoxylinof 5.0 µm in thick and (MonteiroEosin for subsequent& Carvalho 2006, histomorphometric Monteiro et al. describedlected and by the Hafez oestrus & Hafez cycle (2004). stage The were FP classified was characterized in follicular by analysis (Oberlender et al. 2012b). preovulatory follicles (8-11mm in diameter) or almost ovulating Morphometric evaluation (greaterthe presence than of11mm growing in diameter). ovarian follicles In this (2-5mmphase it incan diameter) also be ob or- Histomorphometric analyzes were performed from photomi- served the presence of in regression process and croscopy obtained by the system of image’s capture consisting in without vascularization on the surface, and/or the presence of trinocular microscope (CX31, Olympus Optical do Brasil Ltda, São albicans bodies from previous cycles. On the other hand, LP was Paulo, São Paulo), camera of digital image capture (SC30, Olym- characterized by the presence of recent hemorrhagic bodies or pus Optical do Brasil Ltda, São Paulo, SP), CellB software (Imaging much vascularized corpus luteum in the periphery or in the ovar- Software for Life Science Microscopy, Olympus Optical do Brasil ian stigma. In this phase are also observed small growing follicles (smaller than 2.0mm in diameter) and the presence or absence of was used. After, the obtained images were analyzed with the Ima- corpus luteum. geJLtda, software São Paulo, (NIH, SP) Research and 4× lens Services and 10×Branch, magnification National Institute eyepieces of Mental Health, Bethesda, Maryland, United State of America). Ovaries, uterus and uterine tubes collection and preparation In accordance with Tienthai et al. (2006), microscopic evalua- After slaughter and animals evisceration, the female genita- tion of horns and UTs samples were performed by a single resear- lia, including ovaries, UTs, uterus, , , and uri- - cation, in other words, all the slides were coded before the analysis. polystyrene box with ice (Tummaruk et al. 2009). Subsequently it cherThe (T.P. evaluations Pontelo), who performed had no onknowledge each slide about were: animals total thickness identifi wasnary transported bladder were to collected,the laboratory identified of Physiology and individually and Pharmacolo stored in- gy (DVM/UFLA) within a period of one hour. In order to obtain the ovaries, uterus and UTs of each female, (µm) of the uterine wall (analysis of five different fields in each the reproductive tract was dissected and these structures were normalhistological endometrial sample); glands total thicknessand the dilated of the ones, endometrium when present (µm) (five different fields in each histological sample); diameter of the- and stored in 0.9% (w:v) NaCl solution at 37°C until the morpho- dometrial glandular epithelium and UT epithelial height (20 diffe- logicalremoved and using morphometric scissors. Subsequently analyzes were were performed. adequately identified (20 different fields for each histological sample); height of the en

rent fields for each histological sample). With the use of graticule Pesq. Vet. Bras. 34(1):83-90, janeiro 2014 86 Guilherme Oberlender et al.

- Table 1. Weight and morphometric data (mean±SD) of dometrium occupied by endometrial glands, blood vessels, con- reproductive organs of prepubertal gilts in follicular and containing 352 points was also evaluated the proportion of en luteal stage of oestrus cycle (n = 48 animals) Analyzed Oestrus cycle Mean±SD Minimum Maximum P value nective tissue and epithelium. For this purpose were evaluated 15 variable stage Experimentalfields per animal, design totalizing 5,280 points. For the assessment of morphological, morphometric and his- tomorphometric characteristics of nulliparous gilts ovaries, ute- Weight of the Follicular 36.93±17.44 15.11 76.25 0.203 rus and UTs at different oestrus cycle stages, a simple model of right uterine Luteal 39.36±5.75 35.39 45.95 analysis to compare the mean of two different treatments (Ober- horn (grams) Mean 37.45±15.49 15.11 76.25 lender et al. 2012a) (oestrus cycle stage - FP×LP and the place- Weight of the Follicular 37.00±17.08 15.70 72.94 0.357 ment of reproductive structures - right × left antimer) in the re- left uterine Luteal 40.83±9.28 34.84 51.52 productive tracts obtained were used. horn (grams) Mean 37.82±15.50 15.70 72.94 Total weight Follicular 62.70±32.71 27.86 167.52 0.170 Statistical analysis of the uterus Luteal 54.28±19.15 28.98 97.47 Data are presented as mean±standard deviation (SD). For (grams) Mean 60.42±29.70 27.86 167.52 each analyzed variable (weight of the uterine horns, uterus, uter- Weight of the Follicular 1.01±0.31 0.59 1.62 0.555 ine tubes and ovaries; length of the uterus body, uterine horns, right uterine Luteal 1.22±0.42 0.76 1.59 tube (grams) Mean 1.05±0.33 0.59 1.62 uterine tubes, larger and smaller length of the ovaries, total thick- Weight of the Follicular 1.05±0.33 0.65 1.73 0.851 ness of the uterus and endometrium, endometrial glands diam- Weightleft uterine of the FollicularLuteal 3.72±1.361.13±0.35 0.780.76 7.481.47 0.400 eter, height of the glandular and uterine tube epithelium and tube (grams) Mean 1.07±0.33 0.65 1.73 proportion of endometrium occupied by epithelium, connective tissue glands and blood vessels), data were submitted to normal- Weightright ovary of the FollicularLuteal 4.32±1.234.13±1.85 2.641.85 7.879.38 0.683 ity analysis (Kolmogorov-Smirnov test) and test of the variances (grams) Mean 3.83±1.50 0.78 9.38 homoscedasticity (Levene). Afterwards, data were submitted to Student’s t test to assess the statistical differences between the left ovary Luteal 4.14±1.78 1.93 9.35 two different oestrus cycle stages (FP×LP) and between the place- (grams) Mean 4.27±1.38 1.93 9.35 Length of Follicular 4.17±1.40 3.00 6.50 0.179 ment of reproductive structures (right × left antimer). - the uterus Luteal 5.77±1.17 3.40 5.80 - body (cm) Mean 4.36±1.28 3.00 6.50 ence. All statistical analyses were performed using the statisticalA signifi Length of the Follicular 49.75±12.64 32.50 96.50 0.336 packagecance level SPSS of for5 % Windows was considered version 17.0to indicate. a statistical differ right uterine Luteal 45.71±8.95 30.00 61.50 5 lefthorn uterine (cm) LutealMean 48.66±11.8148.12±7.68 37.0030.00 61.4096.50 RESULTS Length of the Follicular 50.86±11.00 35.00 86.50 0.240 horn (cm) Mean 50.12±10.20 35.00 86.50 FP and 13 gilts (27.08%) in LP of oestrus cycle. Length of the Follicular 18.35±2.35 15.20 22.00 0.052 From a total of animals evaluated, 35 (72.92 %) were in the right uterine Luteal 20.07±4.65 14.70 23.00 lefttube uterine (cm) LutealMean 20.87±3.6318.71±2.85 17.0014.70 24.2023.00 length of the different organs of the gilts reproductive tract Length of the Follicular 20.27±3.18 16.50 26.80 0.947 There was no difference (P>0.05) for the weight and- trus cycle (Table 1). Likewise, regarding the placement of oftube the (cm) right LutealMean 20.40±3.142.72±0.47 16.502.10 26.803.90 reproductiveevaluated in thisstructures, study, classified namely, between in the FP the and right LP and of oes left ovaryLarger (cm) length FollicularMean 2.72±0.372.71±0.32 1.90 3.903.50 0.150 antimer, the weight and length of ovaries, UTs and uterine Smaller length Follicular 1.81±0.27 1.20 2.20 0.198 horns of the gilts, in average and in both oestrus cycle sta- ovary (cm) Mean 1.83±0.26 1.20 2.30 of the right Luteal 1.87±0.25 1.20 2.30 - tricges notvariables showed diameter difference of the (P>0.05) endometrial (Table glands,2, Fig.1). height of Larger length Follicular 2.75±0.33 2.00 3.40 0.446 glandularThere wereepithelium difference and (P<0.05)height of forUT theepithelium histomorphome between of the left Luteal 2.68±0.39 2.10 3.50 ofovary the (cm)left LutealMean 1.94±0.302.73±0.35 1.402.00 2.703.50 Smaller length Follicular 1.92±0.23 1.40 2.50 0.669 gilts classified in the FP and LP of the oestrus cycle (Table phases ovary (cm) of the oestrusMean cycle1.92±0.25 (Table 4).1.40 Gilts in 2.70LP presented compared3). The gilts with classified animals in inFP LP. of Onthe theoestrus other cycle hand, presented the dia- a higher (P<0.05) height of glandular and UT epithelium LP compared with FP of oestrus cycle (Fig.2). Regarding the proportiona higher (P<0.05) of connective proportion tissue of andendometrium blood vessels occupied (Fig.2). by histomorphometricmeter of endometrial analysis, glands was total higher thickness (P<0.05) of the in giltsuterus at glands, whereas animals in FP presented a higher (P<0.05) and endometrium, and proportion of endometrium occu- DISCUSSION This study described the morphology, morphometry and between the animals in FP and LP. histomorphometry of the ovaries, uterus and UTs of prepu- piedFor by theepithelium, proportion no differences of endometrium (P>0.05) occupied were observed by en- bertal gilts in FP and LP of the oestrus cycle. We demons- dometrial tissue, glands and blood vessels, there were di- trated that weight and morphometry of prepubertal gilts reproductive organs does not differ in its placement (right and left antimer) and between the FP and LP of oestrus fference (P<0.05) between the animals classified in both SPSS Statistics 17.0, Rel. 17.0.1. 2008, SPSS Inc, Chicago, IL. cycle. 5

Pesq. Vet. Bras. 34(1):83-90, janeiro 2014 Morphological and morphometric evaluation of prepubertal gilt ovaries, uterine tubes and uterus at different oestrus cycle stages 87

Table 2. Weight and morphometric data (mean ± SD) of reproductive organs of prepubertal gilts in follicular and luteal stage of oestrus cycle in comparison with its placement (right and left antimer) (n = 48 animals) Analyzed Oestrus Placement Mean±SD Minimum Maximum P variable cycle value stage

Weight of the Follicular Left 37.00±17.08 15.70 72.94 0.993 uterine horn Right 36.93±17.44 15.11 76.25 (grams) Luteal Left 40.83±9.28 34.84 51.52 0.275 Right 39.36±5.75 35.39 45.95 Mean Left 37.82±15.50 15.70 72.94 0.877 Right 37.45±15.49 15.11 76.25 Weight of the Follicular Left 1.05±0.33 0.65 1.73 0.716 uterine tube Right 1.01±0.31 0.59 1.62 (grams) Luteal Left 1.13±0.35 0.76 1.47 0.712 Right 1.22±0.42 0.76 1.59 Weight of the FollicularMean Left 4.32±1.231.07±0.33 2.640.65 7.871.73 0.9800.983 ovary (grams) Right 1.05±0.333.72±1.36 0.590.78 1.627.48

Luteal Left 4.14±1.78 1.93 9.35 0.763 Right 4.13±1.85 1.85 9.38 Mean Left 4.27±1.38 1.93 9.35 0.778 Fig.1. Ovaries of prepubertal gilts (A) in follicular Right 3.83±1.50 0.78 9.38 and (B) in luteal stage of oestrus cycle. LO = left Length(cm) of the FollicularLuteal Left 50.86±11.0048.12±7.68 37.0035.00 61.4086.50 0.7340.696 uterine horn Right 49.75±12.64 32.50 96.50 ovary, RO = right ovary, F = follicle and CL = cor- pus luteum. Note the high population of ovarian Right 45.71±8.95 30.00 61.50 follicles in Figure A and the high population of Mean Left 50.12±10.20 35.00 86.50 0.675 CL in Figure B. Right 48.66±11.81 30.00 96.50 Length of the Follicular Left 20.27±3.18 16.50 26.80 0.461 uterine tube Right 18.35±2.35 15.20 22.00 Table 3. Morphometric parameters (mean±SD) of (cm) Luteal Left 20.87±3.63 17.00 24.20 0.507 reproductive organs of prepubertal gilts in follicular and Right 20.07±4.65 14.70 23.00 luteal stage of oestrus cycle (n = 48 animals) Mean Left 20.40±3.14 16.50 26.80 0.898 Analyzed Oestrus Mean±SD Minimum Maximum P value Right 18.71±2.85 14.70 23.00 variables cycle stage Larger length Follicular Left 2.75±0.33 2.00 3.40 0.961 of the ovary Right 2.72±0.472.71±0.32 2.101.90 3.903.50 (cm) Luteal Left 2.68±0.39 2.10 3.50 0.592 Right 2.72±0.37 1.90 3.90 Total thickness Follicular 2183.39±574.87 1359.49 3722.56 0.205 Mean Left 2.73±0.35 2.00 3.50 0.782 of the uterus Luteal 1847.82±352.33 1149.35 2417.77 of the ovary Right 1.81±0.27 1.20 2.20 (µm) Mean 2067.67±527.92 1149.35 3722.56 Smaller(cm) length FollicularLuteal Left 1.94±0.301.92±0.23 1.40 2.702.50 0.4930.291 Total thickness of Follicular 1393.62±385.85 921.76 2238.71 0.221 theEndometrial endometrium FollicularLuteal 1160.18±213.7210.07±3.82 b 914.32 1551.28 glands(µm) diameter LutealMean 1313.12±350.9213.00±10.41 a 914.323.63 2238.7139.87 Right 1.83±0.261.87±0.25 1.20 2.30 5.26 19.04 0.049* Mean Left 1.92±0.25 1.40 2.70 0.588 Height of the Follicular 16.32±2.29 a 12.98 21.79 0.016* (µm) Mean 11.08±6.80b 13.203.63 16.9339.87 We also reported that prepubertal gilts during FP of oestrus cycle presented a higher epithelial and UTs glan- Heightglandular of the FollicularLuteal 15.16±1.0124.77±2.17 a 16.37 28.89 0.034* dular epithelium height, and higher proportion of endome- uterineepithelium tube (µm) LutealMean 18.33±2.2715.92±2.00 b 14.7312.98 20.4921.79 trium occupied by connective tissue and blood vessels. On the other hand, in the LP of oestrus cycle, gilts presented a *Meansepithelium followed (µm) byMean different letters18.57±2.16 in the column14.73 differ28.89 by Student’s t higher diameter of endometrial glands and a higher pro- portion of endometrium occupied by glands. test (P<0.05). Hafez (2004), the FP of oestrus cycle in porcine females is Morphological and morphometric evaluation characterized by the presence of ovaries with follicles in The morphology of ovaries obtained showed, mostly, growth stage and up to nearly ovulating. characteristic of prepubertal animals. In other words, ova- Jiménez Loaiza et al. (2012) studying the relation of ries without the presence of albicans body and corpus lu- physiological status of ovaries with the causes of porcine females discard at different ages, reported, differently of - what were obtained in the present study, that most of fe- teum or ovulation signals (Bagg et al. 2004, Knox 2005) (27.08%)(Fig.1). This stage finding of the can oestrus be confirmed cycle. According by the greater to Hafez num & with FP (34.2%) of oestrus cycle. On the other hand, as ber of gilts classified in the follicular (72.92 %) than luteal males discarded presented in LP (53.1%) in comparison

Pesq. Vet. Bras. 34(1):83-90, janeiro 2014 88 Guilherme Oberlender et al.

Regarding the weight and morphometry of reproductive organs, the obtained values for ovaries weight in both stages of oestrus cycle and in both antimers (right and left) resemble those described by Cowan & MacPherson (1966), Schwarze & Schröder (1970) and McEntee (1990), that demonstrated in their studies ovarian weights ranging from 1.30 to 12.20g, 8.00 to 14.00g and 7.00 to 20.00g respectively. Guimarães et al. (2004) reported average weights of the RO and LO of

were also similar to averages obtained in the present study. The4.40±2.56g results andof ovarian 4.70±7.20g, morphometry, respectively, regardless and these of oestrus values cycle stage and antimer, were similar to those obtained by Bagg et al. (2004), who found an average of 2.41cm for the

from prepubertal gilts of the same breed used in this study. greatestRegarding length the and length 1.59 cmof forUTs, the Schwarze smallest ovary& Schröder length (1970) demonstrated that these structures measure be- - less of oestrus cycle stage and antimer, the values obtained intween the present15.00 and study 30.00cm are within in porcine the physiological females. Thus, range regard for the specie. The values obtained in our study were similar to those reported by Cowan & MacPherson (1966) that des- cribed values of 16.90±2.98cm and 17.73±3.69cm for the length of right and left UT respectively. In this work, as ob- served in our study, the length of UT in the left antimer was numerically greater than the right antimer. Guimarães et al. (2004) working with Landrace gilts, re-

right and left UT length respectively. The data obtained by theseported authors values wereof 45.90±6.80cm higher than those and 35.60±4.29cmobtained in our for study, the Fig.2. Histological section (40×) of prepubertal gilts uterine en- regardless of the oestrus cycle stage, and this fact may be dometrium (A) in follicular and (B) in luteal stage of oestrus cycle. EG = endometrial glands. Note the higher proportion of endometrium occupied by endometrial glands in gilts at luteal animals.due to lower Also, number a possible of animals explanation used would in this be study that (five), the gilts so phase of oestrus cycle in Figure B. used,the findings even being can nulliparous,be justified bycould the already individual have variability reached the of puberty, in contrast to the animals used in the present study Table 4. Proportion of uterine endometrium occupied by that were all prepubertal. According with this explanation, different structures in prepubertal gilts in follicular and luteal stage of oestrus cycle (n = 48 animals) Cowan & MacPherson (1966) described that the length of UTs increases with the advancing of reproductive status. Components of the Oestrus Mean±SD Minimum Maximum P value For the length of uterine horns, regardless of oestrus endometrium cycle stage cycle stage and antimer, the values obtained in our study are in accordance with those described for pigs which are - Epithelium Follicular 4.60±1.18 3.09 6.90 0.457 Luteal 4.16±1.02 a 48.312.95 78.115.87 0.038* sults obtained in the present study differ from those obtai- Mean 4.46±1.13 b 2.95 6.90 nedof 39.80-150.00cm by Guimarães, Machado (Cowan && MacPhersonSantos (2004) 1966). which The repor re- Connective Follicular 65.64±8.55 tissueGlands FollicularLuteal 23.64±9.2460.85±6.89 b 51.96 71.92 and left length of uterine horn, respectively. On the other Mean 64.10±8.25a 48.31 78.11 ted values of 77.20±17.35cm and 90.00±18.09cm for right 5.18 41.52 0.035* hand, our results resemble those obtained by Cowan & Ma- Blood vessels FollicularLuteal 29.95±7.146.12±2.88 a 20.132.99 13.0639.15 0.047* Mean 25.67±9.00b 5.18 41.52 - tively.cPherson In this (1966) work that as well observed as observed values in of our 50.11±10.11cm study for UTs, Luteal 5.04±2.97 3.10 11.95 theand length50.20±10.44cm of uterine for horn right in andleft leftantimer uterine was horns, numerically respec *Means followed by differentMean letters5.77±2.89 in the column2.99 differ13.06 by Student’s t higher than right antimer. Talking about the length of uterine body, the average observedtest (P<0.05). by Diehl et al. (2003), Moreira et al. (2006) and obtained in our study, in FP and LP of the oestrus cycle, Karvelienë & Riðkevièienë (2009), all gilts evaluated in our was similar to demonstrated by Guimarães et al. (2004) study presented active ovaries, so because, the FP and LP of oestrus cycle represent normal cyclicity conditions of Cowan & MacPherson (1966) (2.22±0.83cm) in prepuber- ovaries. tal(4.50±0.28cm), gilts. but higher than the average reported by

Pesq. Vet. Bras. 34(1):83-90, janeiro 2014 Morphological and morphometric evaluation of prepubertal gilt ovaries, uterine tubes and uterus at different oestrus cycle stages 89

Histomorphometric evaluation The results of UTs epithelial height, between oestrus The endometrial glands are tubular structures delinea- cycle stages, obtained in the present study are in accordan- ted by simple columnar epithelium (Monteiro & Carvalho ce with Mayor et al. (2004), which reported that there are 2006) and are presented sinuous and branched. It opens cyclical changes in ciliated and secretory cells of UTs epithe- inwards the endometrium surface, except in the caruncu- lium, and in gilts in FP, the ciliary cells predominate, where- lar areas (in ruminants). According Hafez & Hafez (2004), as in non-pregnant females in LP and in pregnant the secre- during the oestrus, endometrial glands are presented re- tory cells become more apparent that the ciliary epithelium. latively straight and once the increase of progesterone le- vel produced by the corpus luteum under development, its hormones. The estrogen stimulates the growth of ciliated grow up, secrete and become more sinuous and complex. epitheliumFurthermore, and these the synthesis events suffer of granules the influence in secretory of ovarian cells, whereas the progesterone antagonizes the growth of these regression are also observed. - TheThus, glands the begin larger to diameterregress when of endometrial the first signs glands of uterine in the cells.The Therefore, larger proportion this fact justifies of endometrium the higher epithelialoccupied hei by the following reason: the LP of oestrus cycle is characte- ght of UTs in gilts classified in FP of oestrus cycle. rizedgilts classified by a higher in FP concentration compared to of LP progesterone may be explained and this by of oestrus cycle compared with LP (Fig.2) can be explained is secreted by the luteal cells of corpus luteum, placenta dueconnective to the tissuehigher and concentrations blood vessels of in estrogen gilts classified and this, in FPas and adrenal gland (Hafez & Hafez 2004). Furthermore, previously described, is responsible for inducing an incre- progesterone prepares the endometrium for implantation ase in the mass of endometrium and uterine and pregnancy maintenance by increasing the secretory (Hafez & Hafez 2004, Bertan et al. 2006). glands in endometrium and also by the inhibition of myo- metrium motility (Carvalho et al. 2003). According Mayor CONCLUSIONS et al. (2004), uterine endometrium is thicker in pregnant This study described the morphology, morphometry females in comparison with non-pregnant, and this is due and histomorphometry of the ovaries, uterus and UTs of to the morphology of endometrial glands, which varies ac- prepubertal gilts in FP and LP of the oestrus cycle. cording the reproductive stage and undergo hyperplasia The endometrial glands diameter, height of glandular during pregnancy and LP of oestrus cycle. epithelium and of UT epithelium and the proportion of The FP of oestrus cycle in porcine is characterized by endometrium occupied by connective tissue, glands and high estrogen concentrations, and this acts in the uterus blood vessels varies through the oestrus cycle in porcine, inducing an increase in the mass of endometrium and myo- metrium (Hafez & Hafez 2004, Bertan et al. 2006). Thus, The weight and morphometry of prepubertal gilts ova- this fact can explain the higher height of endometrial glan- ries,possibly uterus under and the UTs influence are similar of ovarian in its placement steroids. (right and left antimer) and between the FP and LP of oestrus cycle. compared with LP. dularAccording epithelium to Bertan in gilts et classifiedal. (2006), in during FP of oestrusFP of oestrus cycle Acknowledgements.- To the staff of the slaughterhouse “Matadouro/Fri- cycle, estrogen induce cell proliferation in endometrium, whi- work was supported in part by FAPEMIG (the Research Support Founda- le in LP, higher concentrations of progesterone inhibits the gorífico NUTRILI”, Lavras/MG for supplying the biological samples. This mitosis of endometrial tissue. Progesterone induces endome- and Technological Development), CAPES (Coordination of Improvement of trial stroma differentiation, stimulates glandular secretion in Highertion of theEducation State of Personnel),Minas Gerais), the CNPq Veterinary (the National Science Council Graduate for ProgramScientific- association with accumulation of basal vacuoles in glandular me and the Graduate Directory of the Federal University of Lavras, Brazil. epithelium and promotes the proteins release by endometrial cells, which will help the beginning of embryo development. REFERENCES Regarding the height of UT epithelium, the results ob- Abe H. 1996. The mammalian oviductal epithelium: regional variations in served in the present study are similar to those reported by cytological and functional aspects of the oviductal secretory cells. Histol. Steinbach & Smidt (1970) and Sant’Ana et al. (2003), that Histopathol. 11:743-768. 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