UNIVERSIDADE ESTADUAL DO CEARÁ FACULDADE DE VETERINÁRIA PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS VETERINÁRIAS MESTRADO ACADÊMICO EM CIÊNCIAS VETERINÁRIAS
DUANNY MURINELLY DE SOUZA CUNHA
CARACTERIZAÇÃO SEMINAL E ULTRASSONOGRÁFICA TESTICULAR DE VEADOS-CATINGUEIROS (Mazama gouazoubira)
FORTALEZA – CEARÁ 2019 1
DUANNY MURINELLY DE SOUZA CUNHA
CARACTERIZAÇÃO SEMINAL E ULTRASSONOGRÁFICA TESTICULAR DE VEADOS-CATINGUEIROS (Mazama gouazoubira)
Dissertação apresentada ao Curso de Mestrado Acadêmico em Ciências Veterinárias do programa de Pós-Graduação em Ciências Veterinárias da Universidade Estadual do Ceará, como requisito parcial para a obtenção do título de Mestre em Ciências Veterinárias. Área de Concentração: Reprodução e Sanidade Animal.
Orientador: Prof. Dr. Dárcio Ítalo Alves Teixeira.
FORTALEZA – CEARÁ 2019 2
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Dedico aos meus pais, que sempre me incentivaram aos estudos, meu noivo, pelo apoio de sempre, e aos animais do estudo. 5
AGRADECIMENTOS
A meus pais, Genna e Hernani, por sempre me incentivarem a estudar, não medirem esforços para que tivesse a melhor educação fora e dentro de casa e sempre acreditarem em mim. Ao meu noivo Vitor por sempre ouvir minhas reclamações e dúvidas e acreditar em mim. Aos amigos Maressa, Leonardo, Vilemar, Samara e Fabi pelo apoio, pela ajuda durante várias etapas desses 2 anos e pelos momentos de diversão que aliviaram os estresses desse período. À Bruna, especialmente, que me ensinou tudo de análise seminal, me ajudou com a metodologia do projeto, parceira de experimentos, desabafos, caronas e muito mais. À Mírley por me ensinar ultrassonografia, por sempre ajudar e orientar meu trabalho (mesmo de longe, às vezes) e por todo incentivo durante esses 2 anos. Ao professor Dárcio, que me acolheu com um trabalho em animais selvagens, pela ajuda, orientação e a incansável busca por recursos financeiros, cada vez mais escassos. Aos membros do LADIAR pela solicitude no experimento, conversas leves e encorajamento. Ao Leandro e Samuel pela disponibilidade de contribuir para o projeto e boa vontade em realizar os manejos. Às intituições Ecopoint e Haras Claro por contribuírem com os animais e suas dependências. Ao Herlon, Thalles, Vítor pelo apoio e ajuda no trabalho e por tornarem as viagens de carro mais divertidas, sempre com histórias para contar. Ao prof. Nunes (LTSCO) e à profª Lúcia (LRC) por disponibilizarem dependências de seus laboratórios e equipamentos para realização do experimento. Ao PPGCV e à UECE pela oportunidade de realizar o curso. Ao órgão de fomento à pesquisa FUNCAP (Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico), agência financiadora que tornou possível a realização desse trabalho.
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RESUMO
Em diversas espécies, é reconhecida a importância de técnicas de avaliação da saúde reprodutiva dos machos. Na espécie Mazama gouazoubira, há poucos estudos sobre reprodução. Neste contexto, a avaliação seminal e ultrassonográfica aparecem como importantes ferramentas para analisar o potencial fecundante do macho. Portanto, o objetivo do presente trabalho foi caracterizar a ultrassonografia bidimensional e Doppler testicular e os parâmetros de qualidade seminal, morfometria e ultraestrutura espérmaticas em machos adultos de M. gouazoubira. Foi coletado o sêmen de machos adultos de M. gouazoubira por eletroejaculação (n = 8 ejaculados) e avaliado em cor, aspecto, pH, volume, motilidades total e progressiva, turbilhonamento, vigor, viabilidade, morfologia, morfometria, funcionalidade da membrana, atividade mitocondrial e concentração. Uma alíquota foi separada para análise por microscopia eletrônica de transmissão (MET) e varredura (MEV). Foram mensurados circunferência escrotal e volume testicular. Testículos e epidídimos foram avaliados por ultrassonografia modo B e artéria supratesticular por Triplex Doppler. As imagens bidimensionais foram submetidas à análise computadorizada pelo software ImageJ para determinação do valor numérico de pixels (VNP) e heterogeneidade de pixels (desvio padrão do VNP - DPNP). Baseado na qualidade seminal, os animais foram divididos em 2 grupos (F+ com boa qualidade e F- com baixa qualidade). Os parâmetros de sêmen in natura foram comparados entre três animais diferentes (P < 0,05). Circunferência escrotal, volume testicular, VNP, DPNP e parâmetros de Doppler foram comparados entre os grupos F+ e F- (P < 0,05). Parâmetros de tamanho testicular e de ultrassonografia foram correlacionados (P < 0,05). Experimento 1: Houve diferença entre os indivíduos em relação a funcionalidade de membrana, morfologia e morfometria espermáticas. Os defeitos mais frequentes foram cauda enrolada e peça intermediária encurvada. A análise por MEV demonstraram estruturas perceptíveis apenas por esta técnica, como segmento sub-equatorial e banda serrilhada. A análise por MET revelou características das organelas e composição celular, como a presença de 60 espirais mitocondriais. Experimento 2: O grupo F+ obteve maiores valores de circunferência escrotal, volume testicular, VNP e DPNP testiculares (corte transversal). Não houve diferença entre os grupos nos parâmetros dopplervelociméricos. Houve correlações significativas e positivas entre tamanho testicular (circunferência escrotal e volume testicular) e parâmetros ultrassonográficos (VNP e DPNP testiculares). É possível concluir que diferentes tipos de análise seminal carcaterizam o potencial fértil dos machos de M. gouazoubira. Além disso, as diferenças entre 7 os grupos estão possivelmente ligadas a um desequilíbrio na espermatogênese, sem comprometer o fluxo sanguíneo da artéria supratesticular.
Palavras-chaves: Reprodução. Cervidae. Ultrassom. Biotécnicas seminais.
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ABSTRACT
In multiple species, male breeding soundness evaluation is acknowledged as important techniques. In Mazama gouazoubira, there are few studies on reproduction. So, considering semen and ultrasound evaluation can be an important tool to analyze male fertility potential. This study aims to evaluate fresh semen and to measure scrotal circumference, testicular volume, B mode, and spectral Doppler ultrasound parameters in M. gouazoubira. Semen from adult males was collected using electroejaculation (n = 8 ejaculates) and evaluated based on color, aspect, pH, volume, total and progressive motility, mass movement, vigor, viability, morphology, morphometry, membrane integrity, mitochondrial activity and concentration. One aliquot was used for scanning (SEM) and transmission electron microscopy (TEM). Scrotal circumference and testicular volume measured. Testes and epididymis were evaluated using B mode ultrasound and testicular artery blood flow, using Triplex Doppler ultrasound. B mode images were subjected to computerized analysis using ImageJ, generating numerical pixel value (NPV) and pixel heterogeneity (NPV standard deviation – PSD). The males were divided in two groups (F+ with good seminal quality and F- with poor seminal quality). Fresh semen parameters were compared amongst three males (P < 0,05). Scrotal circumference, testicular volume, NPV, PSD and Doppler parameters were compared between F+ and F- group (P < 0,05). Testicular size and ultrasound parameters were subjected to correlation test (P < 0,05). Experiment 1: There was difference among males regarding membrane integrity, morphology and morphometry. The most frequent defects were simple bent tail and bowed midpiece. Structures only notable using SEM were observed, such as subequatorial segment and serrated band. TEM revealed organelle characteristics and cell composition, for instance the presence of 60 mitochondria spirals. Experiment 2: F+ group had higher scrotal circumference, testicular volume and testes NPV and PSD (cross-sectional view). No difference between the groups was observed in dopplervelocimetric parameters. There were positive significant correlations between testis size (scrotal circumference and testicular volume) and ultrasound parameters (testes NPV and PSD). Thus, different types of semen analysis help in characterizing fertile potential of M. gouazoubira males. Also, the differences between groups are possibly associated to an imbalance on spermatogenesis that does not compromise supratesticular artery blood flow.
Keywords: Reproduction. Cervidae. Ultrasound. Semen analysis.
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LISTA DE ILUSTRAÇÕES
Revisão de literatura
Figura 1 - Machos de veados-catingueiros...... 20
Figura 2 - Radiografia contrastada no corte lateral de testículo direito de touro, mostrando a distribuição da artéria testicular. a – cabeça do epidídimo; b – corpo do epidídimo; c – cauda do epidídimo; 6 – Pars marginalis (da a. testicular); 7- ramos laterais da pars marginalis; 9 – túnica arteriosa testicular; 10 – ramos parenquimatosos...... 23
Quadro 1 - Biometria testicular de cervídeos Mazama gouazoubira...... 22
Capítulo 1
Figure 1 - Photomicrography of gray brocket deer (Mazama gouazoubira) sperm on eosin-nigrosin smears at 1000X magnification. (A): Normal sperm in morphological evaluation. (B): Narrow at base head with proximal droplet (arrow). (C): Short and broad head with proximal droplet (arrow). (D) Detached acrosome and simple bent tail (arrow). (E): Bowed midpiece (arrow). (F): Broken tail (left) and simple bent tail (right). (G): Strongly coiled tail (arrow). (H): Detached normal head. Scale bar = 10 µm……………………………….……………... 57
Figure 2 - Electron microphotography of gray brocket deer (Mazama gouazoubira) sperm in scanning electron microscopy evaluation. (A):
Normal sperm in total visualization with regions identified as the head (H), midpiece (MP), annulus (AN) and principal piece (PP). Scale bar = 10 µm. (B): Normal head presenting apical ridge (AR),
equatorial subsegment (ES) and serrated band (SB). Scale bar = 3 µm (C): Narrow at base head and bent midpiece. Scale bar = 10 µm. (D): Narrow (red arrow) and short broad heads (white arrow). Scale bar =
10 µm. (E): Abnormal head contour. Scale bar = 3 µm (F): Pyriform 10
head. Scale bar = 3 µm. (G): Distal droplet. Scale bar = 10 µm…………………………………………………………………… 58
Figure 3 - Electron microphotography of normal gray brocket deer (Mazama gouazoubira) sperm structures in transmission electron microscopy evaluation. AC: acrosome; AX: axoneme; CAP: capitulum; FS: Fibrous sheath; IAM: inner acrosomal membrane; IF: implantation fossa; MS: mitochondrial sheath; NM: nuclear membrane; NU: nucleus; OAM: outer acrosomal membrane; ODF: outer dense fibers; PAS: post-acrosomal sheath; PC: proximal centriole; PM: plasma membrane; SAS: sub-acrosomal space; SC: segmented columns; red arrows: electron-lucent areas. (A): Sperm head and midpiece. (B): Sperm head in apical region. (C): Post-acrosomal region of the head. (D): Implantation fossa region. (E): Distal midpiece region in longitudinal view. (F): Midpiece and principal piece in cross- sectional view. Numbers 1, 3, 5, 6, and 8 refer to ODF. (G): Principal piece in longitudinal view…………………………………………… 59
Figure 4 - Electron microphotography of gray brocket deer (Mazama gouazoubira) sperm in transmission electron microscopy evaluation.
(A-C): Degenerated sperm. BPM: broken plasma membrane; CR: cytoplasmic residue; DAC: degeneration of acrosome; E: plasma membrane extensions; PM: plasma membrane; S: sub-membranous
swelling. (D-E): Strongly coiled tail……………………………………………………………………. 60
Capítulo 2
Figure 1 - Computer-assisted analysis of the ultrasonographic images of gray brocket deer testes. (A) Position of the transducer for visualizing a longitudinal section of the testis. (B) Position of the transducer for visualizing a cross-sectional section of the testis. (C) Spot-meter technique of pixel analysis: six open squares, 3 × 3 mm2 in size, are placed on a longitudinal section of the ultrasonographic image of the 11
testis. (D) Four open squares, 3 × 3 mm2 in size, are placed on a cross-sectional section of the ultrasonographic image of the testis. (E) Two open squares, 3 × 3 mm2 in size, are placed on a longitudinal section of the ultrasonographic image of the epididymis tail. (F) Total pixel number (Count), mean NPV (Mean), standard deviation of NPV (StdDev), minimum value of NPV (Min), and maximum value of NPV (Max), most frequently occurring NPV within the selection (Mode) shown on a histogram…………………………………………………………… 86
Figure 2 - Mode B ultrasonographic images of right (R) or left (L) epididymis (EPID) and testes (TEST) on longitudinal (LONG) or cross- sectional (CS) views. (A-C) Images of F+ males. (D-F) Images of F- males…………………………………………………………….. 87
Figure 3 - Waveform of supratesticular artery of gray brocket deer using spectral Doppler technique…………………………………………. 88
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LISTA DE TABELAS
Capítulo 1 Table 1 - City, age, weight, proved fertility, housing with female and origin about the three gray brocket deer (Mazama gouazoubira)…………...... 61
Table 2 - General seminal parameters for gray brocket deer (Mazama gouazoubira). Values (Mean ± SD), (n = 8 ejaculates)………………...... 61
Table 3 - Evaluation of mitochondrial activities in gray brocket deer sperm (Mazama gouazoubira). Values (Mean ± SD), (n = 8 ejaculates)………………………………………………………... 62
Table 4 - Morphological evaluation in gray brocket deer sperm (Mazama gouazoubira). Values (Mean ± SD), (n = 8 ejaculates)……………...………………………………………… 63
Table 5 - Sperm morphometrical evaluation in three gray brocket deer (Mazama gouazoubira). Values (Mean ± SD), (n = 8 ejaculates)………………………………………………………... 64
Capítulo 2
Table 1 - City, age, weight, proved fertility, housing with female and origin of
the six gray brocket deer (Mazama gouazoubira)………………… 83 Table 2 - General seminal parameters of normospermic males (F+ group; n = 6 ejaculates) and male 3’s first collection. Values (Mean ± SD).………………...... 83 Table 3 - Testicular parameters of normospermic (F+) and oligo/azoospermic (F-) gray brocket deer (Mazama gouazoubira). Values (Mean ± SD)…………………………….. 83 13
Table 4 - B mode and spectral Doppler ultrasound parameters of normospermic (F+) and oligo/azoospermic (F-) gray brocket deer (Mazama gouazoubira). Values (Mean ± SD)…………………… 84 Table 5 - Correlations between the ultrasound parameters, scrotal circumference, and testicular volume in gray brocket deer (Mazama gouazoubira)…………………………………………. 85
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LISTA DE ABREVIATURAS E SIGLAS
ANOVA Analysis of variance
CAPES Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
CE Circunferência escrotal cm Centímetro/centimeter
CNPq Conselho Nacional de Desenvolvimento Científico e Tecnológico
DAB 3,3’-Diaminobenzidine
DNA Ácido desoxirribonucleico
DPNP Desvio padrão do número de pixels
EDV End diastolic velocity e.g. Exempli gratia
FUNCAP Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico g Grama/gram h Hour
HOST Hypoosmotic test
IM Intrasmuscular
IP Índice pulsatilidade
IR Índice de resistividade
IUCN International Union for Conservation of Nature kg Quilograma/kilogram
MET Microscopia eletrônica de transmissão
MEV Microscopia eletrônica de varredura 15 mg Miligrama/miligram mL Mililitro/mililiter mm Milimetro/milimeter mOsm Miliosmol n number nm Nanômetro/nanometer
NPV Numerical pixel values pH Potencial de hidrogênio/ Hydrogen potential
PI Pulsatility index
PSD Pixel standard deviation
PSV Peak systolic velocity
RI Resistive index
SD Standard deviation s Segundo/second
SEM Scanning electron microscopy
SISBIO System of Authorization and Information on Biodiversity
SNK Student-Newman-Keuls sptz Espermatozoide/sperm
TAMV Time-averaged maximum velocity
TEM Transmission electron microscopy
UFC/CT Universidade Federal do Ceará/ Centro de Tecnologia
UICN União Internacional pela Conservação da Natureza 16
µL Microlitro/microliter
µm Micrometro/micrometer
V Volts
VDF Velocidade diastólica final
VMVP Velocidade média das velocidades de pico
VNP Valores do número de pixels
VPS Velocidade do pico sistólico
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SUMÁRIO
1 INTRODUÇÃO……………………………………………………………. 18
2 REVISÃO DE LITERATURA...... 20
2.1 ASPECTOS GERAIS DO Mazama gouazoubira...... 21
2.2 ANATOMIA REPRODUTIVA DO MACHO CERVÍDEO...... 21
2.3 AVALIAÇÃO REPRODUTIVA DO MACHO...... 23
2.3.1 Avaliação física...... 23
2.3.2 Análise seminal...... 23
2.3.2.1 Parâmetros seminais em M. gouazoubira...... 26
2.3.3 Avaliação ultrassonográfica...... 27
2.3.3.1 Análise computadorizada de imagens ultrassonográficas...... 28
3 JUSTIFICATIVA...... 30
4 HIPÓTESES...... 31
5 OBJETIVOS...... 32
5.1 OBJETIVO GERAL...... 32
5.2 OBJETIVOS ESPECÍFICOS...... 32
6 CAPÍTULO 1...... 33
7 CAPÍTULO 2...... 65
8 CONCLUSÕES...... 89
PERSPECTIVA...... 90
REFERÊNCIAS...... 91
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1 INTRODUÇÃO
O Mazama gouazoubira é um cervídeo autóctone da América do Sul cujo status de conservação global é classificado como pouco preocupante. Todavia, tem apresentado declínio populacional por perda de habitat e caça (BLACK-DÉCIMA; VOGLIOTTI, 2016). Estudos abordando biotécnicas reprodutivas têm sido realizados, caracterizando alguns parâmetros seminais, técnicas de criopreservação e de inseminação artificial (BARROZO et al., 2001; CURSINO; DUARTE, 2016; PERONI, 2013). Os que envolvem ultrassonografia e avaliação seminal são limitados nos cervídeos (GOERITZ et al., 2003; PERONI, 2013). A composição de um tecido ou órgão caracteriza seu aspecto ultrassonográfico. A proporção de água, proteínas e lipídios pode alterar significativamente a ecotextura e ecogenicidade de um órgão ou tecido (SEHGAL, 1993; UNGERFELD; FILA, 2011). Uma gama de informações não visíveis ao olho humano pode ser obtida por meio da análise computadorizada das imagens ultrassonográficas (PIERSON; ADAMS, 1995). Recentemente, moderadas correlações foram encontradas entre parâmetros de imagem (número de pixels) e porcentagem de proteínas e lipídios em testículos de ovinos (AHMADI et al., 2013). A composição bioquímica testicular pode se alterar com a idade e influência hormonal (CONNOR; LIN; NEURINGER, 1997; DERAR; HUSSEIN; ALI, 2012), causando alterações na ecotextura e, assim, ser captada pela análise computadorizada de imagens ultrassonográficas em modo B (UNGERFELD; FILA, 2011). Ademais, a hemodinâmica representa um fator importante para o bom funcionamento da espermatogênese por garantir uma eficiente termorregulação (FOSTER, 2009). A ultrassonografia modo Doppler facilitou o diagnóstico de patologias escrotais como neoplasias, inflamações, traumas e torsões, combinando os achados de ecotextura com a caracterização da perfusão e do fluxo sanguíneo (GÓRECKA-SZYLD, 1999). Além da ultrassonografia, a análise do sêmen é importante na avaliação da saúde reprodutiva do macho, pois sua função é prover um ambiente nutritivo e ionicamente balanceado que contribui para a sobrevivência dos espermatozoides (STABENFELDT; EDQVIST,1996). Fatores nutricionais (BARROZO et al., 2001) e altas temperaturas ambientais (CORCUERA et al., 2002) podem alterar a qualidade seminal, prejudicando a cinética, bem como a morfologia espermática. A análise seminal permite inferir sobre função hormonal, espermatogênese, espermiogênese e maturação espermática (BRITO, 2007; GUR; BREITBART, 2006) e uma análise mais detalhada da morfologia utilizando a microscopia eletrônica permite uma 19 caracterização mais acurada de diferentes estruturas espermáticas (ACKERMAN; REINECKE; ELS, 1996; MEISNER; KLAUS; O’LEARY, 2005). Nesse intuito, trabalhos caracterizando parâmetros seminais e ultrassonográficos têm sido realizados em mamíferos domésticos da ordem Artiodactyla, como bovinos (ARTEAGA; BARTH; BRITO, 2005) e ovinos (AHMADI et al., 2012), bem como em selvagens, como espécies de bovídeos (SANTIAGO-MORENO et al., 2013; WOJTUSIK; WANG; PUKAZHENTHI, 2018) e cervídeos (ROLA; ZANETTI; DUARTE, 2013; UNGERFELD et al., 2017). Dessa forma, pelos escassos estudos acerca da reprodução nos machos de M. gouazoubira (BARROZO et al., 2001; DUARTE; GARCIA, 1995), o presente trabalho tem por finalidade caracterizar a ultrassonografia bidimensional e Doppler testicular e os parâmetros de qualidade seminal, morfometria e ultraestrutura espérmaticas em machos adultos de M. gouazoubira.
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2 REVISÃO DE LITERATURA
2.1 ASPECTOS GERAIS DO Mazama gouazoubira
O M. gouazoubira (veado-catingueiro) pertence à Ordem Artiodactyla (BLACK- DÉCIMA et al., 2010), a qual também compreende as espécies de caprinos (HASSANIN; PASQUET; VIGNE, 1998), ovinos (REZAEI et al., 2010) e bovinos domésticos pertencentes à Família Bovidae (JANECEK et al., 1996). Ela é uma espécie da Família Cervidae cuja ocorrência engloba áreas desde a região ao leste dos Andes, na Argentina e Bolívia até a costa do Oceano Atlântico; e desde o nordeste do Brasil (excluindo a Amazônia) até o sul do Uruguai, indo até a província Entre Rios na Argentina (BLACK-DÉCIMA et al., 2010). O M. gouazoubira é um cervídeo de porte pequeno a médio. Possui uma pelagem de coloração amarronzada, com variações mais claras ou escuras, avermelhadas ou acinzentadas (ROSSI, 2000) (Figura 1). Figura 1 - Machos de veados-catingueiros
Fonte: BLACK-DÉCIMA e colaboradores (2010). Trata-se de um ruminante (ZÚCCARI; SERENO, 2006) e sua alimentação é herbívora, incluindo frutos, plantas lenhosas, gramíneas e ervas (CAVALCANTI; RESENDE; BRITO, 2009; SERBENT;PERIAGO;LEYNAUD, 2011). É um importante dispersor de sementes, contribuindo para composição e distribuição de espécies vegetais (CAVALCANTI; RESENDE; BRITO, 2009). Seus predadores vão desde felídeos selvagens (TABER et al., 1997) a cães ferais (GALETTI; SAZIMA, 2006). Globalmente, é classificado como pouco preocupante pela União Internacional para a Conservação da Natureza (UICN) com declínio populacional devido à caça e perda de habitat (BLACK-DÉCIMA; VOGLIOTTI, 2016). É uma espécie arbustiva e frugívora e, portanto, é mais encontrada em áreas secundárias de bosques pela maior disponibilidade e melhor acessibilidade de alimento de sua preferência (RICHARD; JULIÁ; ACEÑOLAZA, 1995). Possui grande plasticidade ecológica, preferindo áreas remanescentes de florestas e rejeitando áreas abertas, com registros de 21 ocupações em regiões preservadas de vegetação ciliar e Cerrado, bem como em plantações de eucalipto (RODRIGUES; CERVEIRA; DUARTE, 2014; RODRIGUES et al., 2017). Além disso, possui comportamento tímido o que dificulta estudos sobre seu padrão de movimentação (RODRIGUES; CERVEIRA; DUARTE, 2014). São indivíduos de hábitos solitários (BLACK-DÉCIMA, 2000) com maior atividade durante o período diurno (TOBLER;CARRILLO-PERCASTEGUI;POWELL, 2009; FERREGUETTI; TOMÁS; BERGALLO, 2015). Em cativeiro, podem apresentar maior estresse em recintos divididos com outros coespecíficos (CHRISTOFOLETTI; PEREIRA; DUARTE, 2010) e após procedimentos de contenção (MUNERATO et al., 2013). Os machos possuem chifres que surgem, em geral, com a maturidade sexual por volta dos 12 meses, e são renovados anualmente acompanhando mudanças hormonais de gonadotrofinas, esteroides e somatotropina (ZÚCCARI; SERENO, 2006). Baseado na observação do aspecto dos chifres, na presença de fêmeas prenhes ou de recém-nascidos e em avaliações hormonais e seminais, foi constatada a ausência de estacionalidade nos veados- catingueiros (BARROZO et al., 2001; STALLINGS, 1986).
2.2 ANATOMIA REPRODUTIVA DO MACHO CERVÍDEO
O trato reprodutivo dos machos de cervídeo é composto por um par de testículos e epidídimos, ducto deferente, ampola, glândulas acessórias (próstata, glândulas vesiculares, glândulas bulboretrais), pênis, prepúcio (PEREZ; VAZQUEZ; UNGERFELD, 2013; SOHN; KIMURA, 2012) e glândulas prepuciais com função lubrificante e sugerida função imunológica pela presença frequente de plasmócitos (ODEND’HAL; MILLER; DEMARAIS, 1996). Há variações na presença de algumas glândulas acessórias entre as espécies. Por exemplo, na espécie Ozotoceros bezoarticus (veado-campeiro), foi observada a presença de próstata pouco desenvolvida, glândulas vesiculares e a ausência de glândulas bulboretrais (PEREZ; VAZQUEZ; UNGERFELD, 2013), enquanto na espécie Hydropotes inermis argyropus, foram descritas próstata, vesículas seminais e glândulas bulboretrais (SOHN; KIMURA, 2012). Em veado-catingueiro, não há descrições caracterizando suas glândulas acessórias. Até a puberdade, o tamanho testicular aumenta até atingir o da idade adulta (FOSTER, 2009; STABENFELDT; EDQVIST,1996). No M. gouazoubira, o volume testicular médio da espécie (adultos) obtido por Barrozo e colaboradores (2001) foi 32,80 mm3. No trabalho de Costa e colaboradores (2011), os valores médios de biometria testicular observados em sete animais é mostrado no quadro 1. 22
Quadro 1 – Biometria testicular de cervídeos Mazama gouazoubira Parâmetros Mensurações Peso médio do testículo esquerdo (g) 35,6 Peso médio do testículo direito (g) 33,8 Comprimento médio do testículo esquerdo (cm) 6,1 Comprimento médio do testículo direito (cm) 5,9 Largura média do testículo esquerdo (cm) 3,3 Largura média do testículo direito (cm) 3,4 Profundidade média do testículo esquerdo (cm) 3,3 Profundidade média do testículo direito (cm) 3,2 Espessura média da túnica albugínea (µm) 345,7 Volume do parênquima testicular (mL) 64,9 Fonte: Costa e colaboradores (2011).
Adjacente ao testículo, encontra-se o epidídimo, inserido ao longo da borda caudal testicular. A sua cabeça se curva sobre a extremidade dorsal e se estende até a borda cranial. O corpo é situado ao longo da região lateral da borda caudal e a cauda está inserida na região ventral do testículo (SISSON, 1986). No epidídimo, os espermatozoides são armazenados e transportados até o ducto deferente. Durante esse período, ocorre o processo de maturação espermática na qual os gametas masculinos adquirem sua capacidade fecundante (TURNER, 1995). Uma importante região para testículos e epidídimos é o funículo espermático que comporta os vasos sanguíneos (artéria e veia testiculares), formando o plexo pampiniforme, além de vasos linfáticos, nervos e o ducto deferente (SISSON, 1986). Existe um sistema vascular de contracorrente no plexo pampiniforme no qual a natureza pulsátil do fluxo sanguíneo arterial é alterada para formar um sistema contínuo com baixa pressão o que contribui para manter a temperatura testicular e epididimária baixa (FOSTER, 2009). Na região do plexo pampiniforme, a artéria espermática, com seu trajeto tortuoso, segue até o testículo onde se torna a artéria testicular. Atingindo o polo superior do testículo, ela segue para o polo inferior pela região posterior criando ramificações, também, com trajeto tortuoso por toda a superfície testicular e estas seguem para o interior do parênquima. Além disso, existem ramificações para os epidídimos (HARRISON; WEINER, 1949; KHALIFA; NAWAL, 2017) (Figura 2). 23
Figura 2 – Radiografia contrastada no corte lateral de testículo direito de touro, mostrando a distribuição da artéria testicular. a – cabeça do epidídimo; b – corpo do epidídimo; c – cauda do epidídimo; 6 – Pars marginalis (da a. testicular); 7- ramos laterais da pars marginalis; 9 – túnica arteriosa testicular; 10 – ramos parenquimatosos
Fonte: KHALIFA; NAWAL (2017).
Após a espermatogênese, os espermatozoides são transportados para os ductos eferentes através dos túbulos retos, e, depois, para o interior do epidídimo. O caminho para fora da bolsa escrotal é pelo ducto deferente (FOSTER, 2009). A duração do ciclo espermático em cervídeos não é bem elucidada, mas em ovinos e bovinos está entre 60 e 70 dias (DAVIDSON; STABENFELDT, 2008).
2.3 AVALIAÇÃO REPRODUTIVA DO MACHO
2.3.1 Avaliação física
O tamanho, integridade e tonicidade dos testículos são os aspectos mais importantes na avaliação reprodutiva física. Adesões podem ser detectadas por meio de palpação. O volume testicular e a circunferência escrotal (CE) podem ser mensurados e permitem a comparação entre os machos de idades diferentes, férteis e inférteis e em diferentes estações do ano (SPINDLER; WILDT, 2010). Em espécies com estacionalidade, como o Cervus elaphus (HAIGH et al., 1984), Cervus nippon (KAMEYAMA et al., 2000) e Cervus eldi thamin (MONFORT et al., 1993) foi 24 observado um aumento na CE e/ou volume testicular na estação reprodutiva. No Elaphodus cephalophus, todavia, não foram constatadas variações nesses parâmetros em diferentes estações do ano, mesmo com diferenças sazonais nas concentrações séricas de andrógenos e qualidade seminal (PANYABORIBAN et al., 2016). No M. gouazoubira, foi confirmada ausência de estacionalidade reprodutiva (BARROZO et al., 2001; STALLINGS, 1986), entretanto, não há descrições de CE na espécie. Portanto, ressalta-se a importância de haver uma descrição para este parâmetro, pois ele está ligado à função hormonal e à qualidade seminal já que foram observadas moderadas correlações positivas entre concentração de testosterona sérica e as mensurações de CE e volume testicular (KAMEYAMA et al., 2000) e entre as medidas de CE e parâmetros seminais (volume e motilidade progressiva) (RASHID et al., 2015).
2.3.2 Análise seminal
Há uma literatura científica limitada abordando a manipulação da atividade reprodutiva masculina em mamíferos de zoológicos. A maioria dos estudos avaliaram o sêmen ou estabeleceram parâmetros de concentrações hormonais. De modo geral, uma avaliação seminal detalhada inclui a análise de volume, pH, motilidades total e progressiva (subjetivamente ou por análise computadorizada) e vigor, concentração e morfologia dos espermatozoides (SPINDLER; WILDT, 2010). Fatores que podem alterar a qualidade seminal são temperaturas na bolsa escrotal muito baixas ou altas (ARTEAGA; BARTH; BRITO, 2005; JOHNSON, 1997), distúrbios no ambiente testicular e durante o trânsito epididimário (CORREA-PÉREZ et al., 2004), fatores nutricionais (como deficiências em vitaminas A e E, ácidos graxos essenciais, zinco e alguns aminoácidos), distúrbios hormonais, alguns medicamentos, inflamações, infecções (JOHNSON, 1997) e a própria idade do animal (BRITO et al., 2012). A mensuração do volume seminal é importante, pois suas alterações podem indicar obstruções, anomalias anatômicas (VON ECKARDSTEIN et al., 2000), deficiência de andrógenos, ejaculação retrógrada parcial ou indícios de doença inflamatória nas glândulas acessórias (WHO, 2010). A acidez é outro parâmetro importante, pois um sêmen com pH mais ácido pode estar relacionado a processos obstrutivos ou ausência de ducto deferente (VON ECKARDSTEIN et al., 2000). Já um pH mais básico no sêmen de ungulados pode ser causa de contaminação por urina (SPINDLER; WILDT, 2010). 25
Com relação à cinética espermática, maiores valores de motilidade e velocidade dos espermatozoides estão relacionados positivamente a melhores taxas de gestação (LUZ; NEVES; GONÇALVES, 2000; NETO et al., 2012), sendo, portanto, importantes índices de avaliação da saúde reprodutiva. O vigor também é um parâmetro com boa correlação positiva com taxas de prenhez (NETO et al., 2012). A concentração espermática está negativamente relacionada com o tempo para a concepção (SLAMA et al., 2002), sendo um importante preditor para ocorrência de gestação (LARSEN et al., 2000). A morfologia espermática é essencial na análise seminal, pois a porcentagem de defeitos maiores pode ser correlacionada negativamente com melhores taxas de prenhez (NETO et al., 2012), além de ser um importante parâmetro para predizer a capacidade fecundante do sêmen, sendo muito utilizada em procedimento de escolha de amostras para inseminação artificial em ovinos, juntamente com a motilidade progressiva (LUZ; NEVES; GONÇALVES, 2000). Para uma caracterização espermática mais detalhada, pode-se utilizar a microscopia eletrônica. O modo de varredura possibilita uma melhor observação do formato da cabeça e acrossoma e do aspecto da superfície da célula, já com o modo de transmissão, pode-se descrever a morfologia interna da célula espermática, definindo núcleo, acrossoma, espaço subacrossomal, bainha mitocondrial e axonema (SILVA et al., 2015). Estudos utilizando MET têm sido realizados para caracterização morfológica (BARTOOV et al., 1980), efeitos de diferentes diluentes (BADR et al., 2015) e dietas (YOUNAN; EL-GABRY; MORSY, 2017) nos espermatozoides. A mensuração morfométrica é importante, pois características como uma cabeça mais alongada (juntamente com uma velocidade mais rápida e linear) em maior porcentagem está correlacionada com uma melhor fertilidade (RAMÓN et al., 2013). Outras avaliações essenciais são os testes de viabilidade e funcionalidade de membrana, capazes de detectar células com lesão em sua membrana e células com alteração na permeabilidade desta, respectivamente. Além disso, uma maior quantidade de células viáveis no teste de viabilidade pode estar correlacionada com maiores taxas de prenhez (BARROS et al., 2007). E, a partir do trabalho de Siqueira e colaboradores (2007), pode-se sugerir que células com membrana funcional têm mais resistência térmica (temperatura de 37º C). A atividade mitocondrial das células espermáticas pode ser avaliada utilizando o composto 3-3’diaminobenzidino. Células com alta atividade mitocondrial podem estar correlacionadas com melhor motilidade e funcionalidade de membrana (BARROS et al., 2007), 26 importantes resultados para obtenção de melhores taxas de prenhez no rebanho (LUZ; NEVES; GONÇALVES, 2000; NETO et al., 2012; SIQUEIRA et al., 2007). Diante do exposto, ressalta-se que utilizar diferentes testes para a avaliação funcional dos espermatozoides é importante, pois fornece uma melhor previsibilidade do potencial fértil da amostra analisada (BARROS et al., 2007). O sêmen de algumas espécies de cervídeos tem sido estudado há algumas décadas. Exemplificando, na espécie C. elaphus, foram realizadas análises morfológicas e de viabilidade do sêmen fresco (HAIGH et al., 1984), enquanto no sêmen do C. eldi thamin, volume, motilidades total e progressiva e morfologia foram descritos (MONFORT et al., 1993). No C. elaphus, foram avaliados pH, aspectos físicos, volume, motilidade progressiva, morfologia e concentração (GIŻEJEWSKI, 2004). Na espécie Dama dama, avaliou-se motilidade progressiva, vigor, funcionalidade da membrana e integridade acrossomal (FERNÁNDEZ et al., 2013). Na espécie Ozotocerus bezoarticus, caracterizou-se cor, pH, motilidades, volume, concentração, integridade de membrana e morfometria espermética (BERACOCHEA et al., 2014). Além dessas análises, em avaliações por microscopia eletrônica de varredura, os espermatozoides dos cervídeos D. dama (GOSCH; BARTOLOMAEUS; FISCHER, 1989), Axis axis, C. eldi, C. nippon taivunus, Odocoileus virginianus (MEISNER; KLAUS; O’LEARY, 2005) e Rusa timorensis foram caracterizados (MAHRE et al., 2014). Ademais, no gênero Mazama sp., descrições de volume, pH, aspectos físicos, osmolaridade, turbilhonamento, motilidades, vigor, concentração, morfologia, morfometria (ROLA; ZANETTI; DUARTE, 2013), volume e integridade de membrana do sêmen de M. americana foram relatadas (FAVORETTO; ZANETTI; DUARTE, 2012). Volume, concentração e morfometria foram caracterizados em M. nemorivaga (CURSINO; DUARTE, 2016), enquanto, em M. nana, foram avaliados características físicas, volume, motilidade progressiva, vigor, morfologia e concentração do ejaculado (ABREU et al., 2009).
2.3.2.1 Parâmetros seminais em M. gouazoubira
Estudos caracterizando parâmetros seminais em M. gouazoubira ainda são preliminares e, como não existem valores de referência para a espécie, utilizam-se as classificações para pequenos ruminantes (CBRA, 2013). Nessa espécie, têm sido descritos alguns parâmetros seminais. 27
O volume médio do ejaculado variou entre 0,1 a 0,7 mL, em coleta por eletroejaculação (CURSINO; DUARTE, 2016; DUARTE; GARCIA, 1995). No trabalho de Peroni (2013), a motilidade total do sêmen fresco foi 80%, caindo para 40% a 60% pós-descongelação. O vigor do sêmen fresco, foi de 4, enquanto, após descongelação, foi de 1 a 3. Estudos anteriores analisando a morfologia espermática encontraram porcentagens de 42% a 57% de células com defeitos em amostras de sêmen fresco e de 51% a 82%, após descongelação, em coleta por eletroejaculação (PERONI, 2013). Além disso, estudos com morfometria relataram médias de 7,13 a 8,53 µm para o comprimento da cabeça; 50,24 a 53,04 µm para o comprimento da cauda e 10,67 a 11,63 µm para comprimento da peça intermediária (CURSINO; DUARTE, 2016). Ademais, a viabilidade espermática encontrada, utilizando os corantes eosina- nigrosina, variou de 67% a 78% (PERONI, 2013). A concentração seminal variou de 0,85 a 3,9 x 109 espermatozoides por mililitro no trabalho de Cursino (2014). Outros parâmetros foram descritos, como a integridade do acrossoma (34% a 62%) e o índice de fragmentação de DNA (0,3% a 0,7%) do sêmen descongelado. Além desses, o teste de termorresistência foi realizado, resultando em avaliações de 31% a 44% de motilidade e 1,5 a 2,2 de vigor (PERONI, 2013).
2.3.3 Avaliação ultrassonográfica
Assim como a análise seminal, a ultrassonografia é uma ferramenta importante na avaliação reprodutiva. Ela tem sido usada em estudos sobre topografia anatômica, conhecimento essencial para desenvolver técnicas de coleta de sêmen por manipulação ou eletroejaculação (ZÚCCARI; SERENO, 2006), e para estabelecer parâmetros de normalidade em estágios pré e pós-puberdade em animais sem afecções (DERAR; HUSSEIN; ALI, 2012). Além disso, ela tem sido utilizada em pesquisas sobre a caracterização da puberdade, estágios do processo ejaculatório, mudanças sazonais nos testículos (ZÚCCARI; SERENO, 2006), avaliações do trato reprodutor, realizando mensurações das dimensões testiculares e das glândulas acessórias (CAMELA et al., 2017; GOERITZ et al., 2003), auxiliando, assim, no monitoramento do manejo reprodutivo (ZÚCCARI; SERENO, 2006) e na detecção de animais subférteis ou inférteis a partir do diagnóstico de certas alterações no parênquima testicular (ORTIZ-RODRIGUEZ et al., 2017). 28
Essa técnica contribui para a caracterização de patologias no trato reprodutor masculino ao especificar a localização e o tipo de alteração observada no órgão, ajudando a definir o prognóstico. Patologias como orquite, epididimite, cistos, degeneração testicular, hematomas, abscessos, hidrocele, hipoplasia testicular e neoplasias podem ser detectadas no exame ultrassonográfico em modo B (GNEMMI; LEFEBVRE, 2009). Além do modo B, o uso da técnica Doppler é uma ferramenta igualmente importante da ultrassonografia por possibilitar a avaliação da perfusão dos tecidos através da análise da arquitetura vascular, da direção do fluxo e da sua velocidade. Doenças inflamatórias podem não ser detectadas no modo B, mas alterações no índice de resistividade (índice dopplervelocimétrico) do fluxo arterial testicular apresentaram uma alta sensibilidade e especificidade no diagnóstico desses processos em homens (JEE et al., 1997), sugerindo que alterações na resistência vascular e, consequentemente, no fluxo sanguíneo, interferem na espermatogênese (PINGGERA et al., 2008). Além disso, importantes correlações entre parâmetros dopplervelocimétricos e qualidade seminal, estradiol-17β e volume testicular foram descritos em ovinos (HEDIA et al., 2019). Portanto, a elaboração de valores de referência para os parâmetros dopplerfluxométricos da artéria testicular tem grande importância para o estudo da vascularização testicular e diagnóstico de patologias (POZOR; MCDONNELL, 2004).
2.3.3.1 Análise computadorizada de imagens ultrassonográficas
A análise computadorizada das imagens ultrassonográficas é uma técnica com muitas aplicações na pesquisa a qual tem sido utilizada para estudar alterações causadas por altas temperaturas na bolsa escrotal (insulação) (ARTEAGA; BARTH; BRITO, 2005; BRITO et al., 2003), mudanças testiculares relacionadas às fases pré e pós-pubertal (BRITO et al., 2012) e ocorridas durantes estações reprodutiva e não reprodutiva (AHMADI et al., 2012). Além disso, estudos demonstram o potencial da análise de pixels nas imagens como um fator preditor da qualidade seminal (AHMADI et al., 2012; ARTEAGA; BARTH; BRITO, 2005). Em trabalhos posteriores, foram observadas correlações positivas, não só entre, a área ocupada pelos túbulos seminíferos e os números de pixels, como também, entre a área de lúmen dos túbulos e a heterogeneidade de pixels em ovinos (GIFFIN et al., 2009; GIFFIN;BARTLEWSKI; HAHNEL, 2014). Ademais, foram encontradas correlações entre intensidade de pixels e porcentagens de proteína, lipídios e água (AHMADI et al., 2013). 29
Portanto, ela pode ajudar a identificar alterações na ecotextura ultrassonográfica mais objetivamente (PIERSON; ADAMS, 1995). A literatura sobre ultrassonografia do trato reprodutivo masculino em cervídeos é escassa. Na espécie Capreolus capreolus, foram avaliados testículos, epidídimos e glândulas acessórias no modo bidimensional (GOERITZ et al., 2003), enquanto, no O. bezoarticus, foram avaliados os testículos no mesmo modo e obtidos os valores de intensidade de pixel (UNGERFELD et al., 2017).
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3 JUSTIFICATIVA
Estudos com biologia reprodutiva e técnicas de reprodução assistida em animais selvagens têm sido realizados com objetivo de melhorar o manejo e a reprodução em cativeiro (BERNDT, 2005; LEITE et al., 2006; SILVA et al., 2015; OLIVEIRA et al., 2016). Estudos do sêmen de veado-catingueiro são preliminares (CURSINO; DUARTE, 2016; ROLA; ZANETTI; DUARTE, 2012) e, aliado à ultrassonografia, pode fornecer informações importantes sobre a capacidade reprodutiva do animal, possibilitando inferir sobre o potencial fértil espermático (PERONI, 2013), analisar o parênquima dos testículos e epidídimos (AHMADI et al., 2013) e avaliar fluxo sanguíneo e perfusão nos mesmos (LAM et al., 2005; POZOR; MCDONNELL, 2004). Os conhecimentos sobre reprodução e técnicas de análise da capacidade reprodutiva são a base para o desenvolvimento de eficazes programas de reprodução (OLIVEIRA et al., 2016), pois o conhecimento sobre características reprodutivas e técnicas que auxiliem o diagnóstico de patologias nos machos dessa espécie contribuem para o desenvolvimento de biotécnicas reprodutivas e permitem uma seleção dos machos mais saudáveis em programas de reprodução assistida (ORTIZ-RODRIGUEZ et al., 2017), garantindo um manejo reprodutivo bem-sucedido. Apesar do M. gouazoubira ser classificado globalmente como “pouco preocupante”, ele é ameaçado pela caça, por doenças transmitidas por animais domésticos e pela perda e destruição do seu habitat (DELLAFIORE, MACEIRA, 2001) e, pela proximidade filogenética com as espécies M. bororo e M. nana (ameaçados de extinção globalmente), ele, também, pode ser uma espécie modelo para desenvolvimento de biotécnicas seminais para esses cervídeos brasileiros (BLACK-DÉCIMA; VOGLIOTTI, 2016). Portanto, essa pesquisa torna-se válida em M. gouazoubira, por se tratar de uma espécie com poucos estudos em reprodução e possuir populações de vida livre em declínio; ser modelo para espécies ameaçadas globalmente; e, ainda assim, haver disponibilidade de indivíduos em cativeiro.
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4 HIPÓTESES
Há diferença significativa em parâmetros ultrassonográficos, de circunferência escrotal e volume testicular em animais com boa e baixa qualidade seminal.
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5 OBJETIVOS
5.1 OBJETIVO GERAL - Avaliar a qualidade de espermatozoides a fresco (incluindo avaliação morfométrica e ultrasestrutural) e comparar os parâmetros ultrassonográficos bidimensionais (intensidade e heterogeneidade de pixels) dos testículos e dopplervelocimétricos da artéria testicular em veados Mazama gouazoubira com boa e baixa qualidade seminal.
5.2 OBJETIVOS ESPECÍFICOS - Descrever os parâmetros seminais (in natura) de motilidade total e progressiva, vigor, turbilhonamento, volume, concentração, viabilidade, funcionalidade de membrana e atividade mitocondrial na espécie; - Descrever a morfometria espermática e ultraestrutura dos espermatozoides da espécie; - Verificar se há correlações entre os parâmetros ultrassonográficos (intensidade e heterogeneidade de pixels e de Doppler espectral) e de tamanho testicular (circunferência escrotal e volume testicular) na espécie.
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6 CAPÍTULO 1
Caracterização dos parâmetros seminais, morfometria e ultraestrutura dos espermatozoides de veado-catingueiro (Mazama gouazoubira, Fischer, 1814)
Characterization of seminal parameters, sperm morphometry, and ultrastructure in gray brocket deer (Mazama gouazoubira, Fischer, 1814)
Duanny Murinelly de Souza Cunha, Mírley Barbosa de Souza, Bruna Farias Brito, Herlon Victor Rodrigues Silva, Leandro Rodrigues Ribeiro, Francisco Antônio Félix Xavier Júnior, Janaina Serra Azul Monteiro Evangelista, Leda Maria Costa Pereira, Dárcio Ítalo Alves Teixeira
Periódico: Microscopy Research & Technique (Submetido em 10 de julho de 2019) Qualis B1 34
1 Characterization of seminal parameters, sperm morphometry, and ultrastructure in 2 gray brocket deer (Mazama gouazoubira, Fischer, 1814) 3 4 Semen features in gray brocket deer 5 6 Duanny Murinelly de Souza Cunhaa, Mírley Barbosa de Souzaa, Bruna Farias Britoa, Herlon 7 Victor Rodrigues Silvaa, Leandro Rodrigues Ribeirob, Francisco Antônio Félix Xavier Júniora, 8 Janaina Serra Azul Monteiro Evangelistaa, Leda Maria Costa Pereiraa, Dárcio Ítalo Alves 9 Teixeiraa* 10 11 a Faculty of Veterinary Medicine, Ceara State University, Fortaleza, Brazil. 12 b Claro Commercial Wild Animal Breeding Farm, Caucaia, Brazil. 13 14 *Corresponding author: Dárcio Ítalo Alves Teixeira 15 E-mail address: [email protected] 16 Phone: +55 85 31019850 35
17 ABSTRACT 18 Populations of gray brocket deer (Mazama gouazoubira) are declining; yet, knowledge on the 19 reproductive biology of this species remains limited. Therefore, this study aimed to describe 20 the sperm cells of gray brocket deer to obtain baseline information in the reproductive 21 characteristics of male semen and develop reproductive biotechniques. Three adult male gray 22 brocket deer were used in the study. Semen collection was performed using electroejaculation. 23 Semen were analyzed by evaluating pH, motilities, vigor, mass movement, volume, 24 concentration, viability, membrane integrity, mitochondrial activity, morphology and 25 morphometry. Ultrastructure of sperm was analyzed using scanning and transmission electron 26 microscopy (SEM and TEM). There was no significant difference among males regarding on 27 pH, motilities, vigor, mass movement, volume, concentration, viability. High values for 28 membrane integrity, mitochondrial activity and normal sperm were observed. The most 29 frequent defects were simple bent tail and bowed midpiece. The head length, and width, 30 midpiece, and tail length were 8.5, 4.4, 11.5 and 41.3 µm, respectively. SEM sperm showed 31 paddle-shaped heads, with apical ridge and serrated band on the equatorial segment. TEM 32 revealed the nucleus, acrosome, plasma membrane, mitochondria sheath, proximal centrioles, 33 segmented columns, axoneme, outer dense fibers and fibrous sheath. SEM and TEM showed 34 the presence of some abnormalities. These results are expected to improve male selection, 35 contributing towards the development of reproductive biotechnologies for gray brocket deer 36 and, other deer species at risk of extinction. 37 38 Keywords: Gamete, Wild animal, Cervidae, HOST, DAB, Electron microscopy. 39 40 41 Highlights 42 43 • All males presented high kinetic parameters, viability and membrane integrity. 44 • All males had high mitochondria activity and animal 1 presented lower DAB 3. 45 • All animals presented good morphology, but animal 1 had higher minor abnormalities. 46 • Ultrastructure analysis revealed a paddle-shaped head and 60 mitochondrial spirals. 47 48 49 36
50 1. INTRODUCTION 51 52 The gray brocket deer (Mazama gouazoubira) is an endemic species from South 53 America that is found in most regions of Brazil (except the Amazon Forest), Uruguay, Paraguay 54 and Bolivia (Black-Decima & Vogliotti, 2016). This species is an important seed disperser 55 (Cavalcanti, Resende, & Brito, 2009), and contributes to the food chain as a prey for carnivores, 56 such as wild felids (Taber, Novaro, Neris, & Colman, 1997). The global conservation status of 57 M. gouazoubira is classified as of “least concern”, according to the International Union for 58 Conservation of Nature (IUCN) Red List. However, this species has been subject to population 59 declines, due to the loss of habitat and hunting (Black-Decima & Vogliotti, 2016). Yet, few 60 studies have investigated fresh semen characteristics, or sperm morphometrics in M. 61 gouazoubira. Existing studies have assessed testicular biometry and histology (Costa et al., 62 2011), semen collection (Rola, Zanetti, & Duarte, 2012), sperm analysis (Barrozo, Toniollo, 63 Duarte, Pinho, & Oliveira, 2001; Cursino & Duarte, 2016) and artificial insemination 64 techniques (Duarte & Garcia, 1995). Sperm morphometry has only been described to 65 differentiate from other Mazama sp. (Cursino & Duarte, 2016). 66 Yet, studies on semen characteristics are important to obtain baseline information on 67 spermatogenesis and spermiogenesis, along with the incidence of certain abnormalities 68 (Bartoov, Eltes, Weissenberg, & Lunenfeld, 1980; Cooper & Yeung, 2003). Such knowledge 69 facilitates the correct diagnosis of issues, allowing males with greater fertility potential to be 70 selected in assisted reproduction programs (Love, 2011). Also, semen analysis contributes to 71 the creation of biobanks that preserve genetic material (Comizzoli, 2015). Ultrastructural and 72 morphometric studies are important for establishing phylogenetic relationships (Cursino & 73 Duarte, 2016; Meisner, Klaus, & O’Leary, 2005). Furthermore, electron microscopy allows 74 sperm cell to assessed in greater detail, overcoming, conventional light microscopy limitations. 75 Transmission electron microscopy (TEM) is used to detect sperm abnormalities with high 76 accuracy, especially in the acrosome, which might have an important effect on fertility (Mundy, 77 Ryder, & Edmonds, 1994; Pesch, Bostedt, Failing, & Bergmann, 2006). 78 Thus, gray brocket deer could be used as a model to develop reproductive 79 biotechnologies for other Mazama sp. considered vulnerable, such as M. bororo and M. nana 80 (Black-Decima & Vogliotti, 2016). 81 There are no previous descriptions of gray brocket deer sperm using hypoosmotic tests, 82 mitochondrial activity, scanning and transmission electron microscopy. Therefore, this study 83 aims at increasing the knowledge about gray brocket deer semen and provide information about 37
84 the different regions of the cell, including sperm morphometry and ultrastructure using scanning 85 and transmission electron microscopy. 86 87 2. MATERIAL AND METHODS 88 89 2.1. Animals 90 91 The study was approved by Ceara State University Ethics Committee (nº 7913746- 92 2017) and by the System of Authorization and Information on Biodiversity – SISBIO (nº 93 60925). 94 Three adult male gray brocket deer (M. gouazoubira) were used in the study. The 95 animals were housed in private institutions; two were at Aba-Yby Institute – Ecopoint 96 Environmental Education Ltd., Fortaleza, CE, Brazil (3°45'57.8"S, 38°34'41.2"W) and one was 97 at Claro Commercial Wild Animal Breeding Farm, Caucaia, CE, Brazil (3°50'57.8"S, 98 38°51'57.3"W). The animals were subjected to the usual management of practices of the 99 respective institutions. At Claro Farm, the animals were fed equine food, grass (Cybodon 100 dactylon and Brachiaria sp.) and roots (e.g. carrots and yuca) daily. At Aba-Yby Institute, 101 animals were offered ovine food, fruits and grass daily. At both institutions, the animals 102 received water ad libitum. More information about the animals is provided in Table 1. 103 104 2.2. Semen collection 105 106 Semen were collected on three occasions from animals 2 and 3 from January to 107 December 2018. Semen were collected on two occasions from animal 1, because it died 108 unexpectedly due to territorial dispute with animal 3. Therefore, eight samples (n = 8 ejaculates) 109 were collected in total at 2.5 to 8 month intervals. Before all procedures, all animals were 110 sedated using 5 to 10 mg/kg ketamine hydrochloride (Cetamin, Syntec, Brazil) and 0.5 to 1.5 111 mg/kg xylazine hydrochloride (Xilazina 10%, Venco, Brazil), both of which were administrated 112 IM (Cursino & Duarte, 2016; Duarte & Garcia, 1995). 113 The animals were positioned in lateral decubitus. The penis and prepuce were cleaned 114 with saline solution (sodium chloride 0.9%) and excess feces were removed from the rectum 115 before introducing the probe with the electrodes positioned towards the prostatic surface. 116 Semen was collected using an electroejaculator (Neovet Autoejac v2, Neovet, Uberaba, Brazil). 117 The procedure consisted of three sets of stimuli, with 10 stimuli for each voltage. The first series 38
118 started with 4 V, 5 V, and 6 V; the second had 5 V, 6 V, and 7 V; and the third had 6 V, 7 V, 119 and 8 V. Each series was separated by a 5 minute interval rest (Monfort et al., 1993). Semen 120 was collected in a 50 mL conical tube. Four aliquots from each sample (collection) were 121 separated for fresh semen analysis, and scanning and transmission electron microscopy. 122 123 2.3. Semen evaluation 124 125 Initially, the semen color, aspect and volume were evaluated. The pH was analyzed 126 using pH measuring sticks (model K36-014, Kasvi, China) (Rola, Zanetti, & Duarte, 2013). 127 The microscopic parameters of total and progressive motilities and vigor (score 0 to 5) were 128 assessed using a light microscope at 100X magnification. The mass movement (score 0 to 5) 129 was analyzed under a light microscope at 40X magnification using a 10 µL sample of fresh 130 semen on a slide. 131 A 10 µL fresh semen aliquot was added to a solution containing 2 mL 1% formaldehyde- 132 saline (0.9% sodium chloride solution). The concentration of sperm was quantified by counting 133 the cells in five fields in a Neubauer chamber with a light microscope at 400X magnification 134 (Rola et al., 2013). 135 Eosin-nigrosin stained smears (Baril et al., 1993) were prepared to evaluate sperm 136 viability and morphology. Viability was quantified by analyzing 200 cells per smear with a light 137 microscope at 400X magnification. Morphology was quantified by analyzing 200 cells per 138 smear with a light microscope at 1000X magnification with immersion oil. The percentages of 139 normal cells and, cells with minor and major defects were determined (Blom, 1972). 140 The integrity of the plasma membrane was evaluated using the hypoosmotic test 141 (HOST). This test involved a adding 10 µL fresh semen to 100 µL of hypoosmotic solution 142 (7.35g sodium citrate, 13.5g fructose and 1000mL distilled water; 100 mOsm/L), and 143 incubating it in a water bath at 37 ºC for 1 h. Afterwards, a slide with a coverslip was prepared, 144 and 200 cells were analyzed using a light microscope at 400X magnification. The percentage 145 of reactive cells (with tail edema or bent tail) was obtained, after subtracting the number of bent 146 and coiled tails observed in the morphological analysis (Oliveira et al., 2013). 147 Mitochondrial activity was assessed by adding 10 µL fresh semen to 25 µL 3,3'- 148 diaminobenzidine (DAB) solution (0.0045mg DAB and 300 µL phosphate buffered saline - 149 PBS). The dilution was kept in a water bath at 37 ºC for 40 min, and was protected from the 150 light. Subsequently, slides were prepared and fixed in 10% formaldehyde solution during 10 151 minutes with light isolation. Then, the slides were rinsed with distilled water, dried at ambient 39
152 temperature (approximately 27 ºC), and analyzed under a contrast phase microscope at 400X 153 magnification. Then, 200 cells were evaluated and classified according to midpiece staining as 154 DAB 1 (100% stained), DAB 2 (more than 50% stained), DAB 3 (less than 50% stained), and 155 DAB 4 (no staining). (Hrudka, 1987). 156 157 2.4. Analysis of sperm morphometry 158 159 For the morphometric analysis, the same slides were used as those in the morphological 160 evaluation (n = 6 ejaculates). Two hundred normal sperm cells were assessed under a light 161 microscope (400x of magnification) connected to a camera. The images were captured using 162 the image analyzer software NIS – Elements (Nikon, Japan). Sperm structure was measured 163 (µm) using image analysis software (ImageJ: Wayne Rasband - National Institute of Health, 164 Maryland, United States); measurements were made of head length and width, midpiece length, 165 and tail length (Fig. 1A) (Sousa et al., 2013). 166 167 2.5. Scanning electron microscopy 168 169 For the scanning electron microscopy (SEM), 20 µL samples were fixed in 2.5% 170 glutaraldehyde in 0.01M phosphate buffered saline (pH 7.4 at 25 ºC). The protocol followed 171 the guidelines of the Central Analítica Laboratory (Physics Department, Ceara Federal 172 University, Fortaleza, Brazil), except for dehydration. Aliquots were placed on round coverslips 173 with poly-d-lysine (Sigma-Aldrich, Cotia, Brazil). Then, they were dehydrated in a series of 174 different concentrations of ethanol (20%, 50%, 80%, 95%, and twice in 100%) (Meisner et al., 175 2005), and critical point dried (EMS 850, Quorum Technologies, Lewes, United Kingdom). 176 The following steps were used to assemble the material in a specimen holder (stub) and coat it 177 thinly with silver through ‘sputtering’ to observe in the SEM (Quanta 450-FEG, Thermo Fisher 178 Scientific, Massachusetts, United States). 179 180 2.6. Transmission electron microscopy 181 182 For TEM, 60 µL samples were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde 183 in 0.2M phosphate buffer (pH 7.4). The protocol followed the guidelines of the Central 184 Analítica Laboratory (Physics Department, Ceara Federal University, Fortaleza, Brazil). The 185 fixed samples were washed three times in phosphate buffered solution. Then, the samples were 40
186 post-fixed with osmium tetroxide for 1 h and washed three times in phosphate buffered solution. 187 Subsequently, the samples were dehydrated in a series of acetone (50%, 70%, 90%, and three 188 times in 100%), and were embedded in Epon resin (Embed 812, Electron Microscopy Sciences, 189 Hatfield, United States). Ultrathin sections (50 nm) were manually stained with uranyl acetate 190 and lead citrate. Different fields were randomly selected, evaluated by TEM (Jeol JEM-1011, 191 Japan), and photographed for later analysis and description. 192 193 2.7. Statistical analysis 194 195 The data were analyzed using the statistical software R-project© (The R Foundation, 196 Vienna, Austria). The data were subjected to the Cramer-Von Mises normality test and the Box- 197 Cox homoscedasticity test. For comparison of means, the homogenous and normally distributed 198 parameters (pH, volume, total motility, progressive motility, concentration, viability, 199 membrane integrity, mitochondrial activity, normal morphology, major, and minor 200 abnormalities) were subjected to ANOVA one-way followed by Student-Newman-Keuls 201 (SNK) test. Lambda (λ) transformation was performed on membrane integrity, mitochondrial 202 activity DAB 3 and 4, normal sperm and major abnormalities (morphology). The non- 203 parametric data (mass movement, vigor, head length and width, midpiece tail, and total sperm 204 length) were subjected to the Kruskal-Wallis test followed by SNK test. The results are 205 expressed as mean ± SD. Significance was set at P < 0.05. 206 207 3. RESULTS 208 209 3.1. Semen evaluation 210 211 All samples were white in color, ranging from watery to milky in aspect. The largest 212 volume of semen was obtained during the second or third electric stimulus cycle. pH was a 213 mean 6.7 ± 0.5. The total volume of the ejaculate was 298.1 ± 120.5 µL; however, there was 214 wide variation in volume, ranging from 150 to 505 µL in males 1 and 2, respectively. The 215 animals presented homogenous values for total motility (96.7 ± 3.2%), progressive motility 216 (86.9 ± 5.9%), mass movement (4.8 ± 0.2), and vigor (4.4 ± 0.5). Concentration varied greatly 217 (94.9 ± 55.1 x 10⁷ sptz/mL), especially in animals 2 and 3. Samples were more homogenous for 218 the viability values, with a mean of 87.6 ± 4.5%. However, for the membrane integrity 41
219 evaluation, animal 1 had lower (46.7 ± 0.3%) values than the other animals (animal 2: 78.3 ± 220 8.5% and animal 3: 86.2 ± 10.6%). All semen values are presented in Table 2. 221 All animals had high active mitochondria levels (DAB 1: 45.7 ± 22.9%; DAB 2: 35.4 ± 222 14.4%). Animal 1 had lower values for DAB 3 mitochondrial activity (3.75 ± 0.3%) than the 223 other animals (animal 2: 21.8 ± 2.5%; animal 3: 19.2 ± 13.2%). All results on mitochondrial 224 characteristics are presented in Table 3. 225 Normal morphology was observed in 72.7 ± 14.8% of the samples (Fig. 1A), while 226 major abnormalities were observed in 9.4 ± 9.2% of the samples and minor abnormalities in 18 227 ± 15.6% (Table 4). Animal 3 had the highest values for normal sperm (85.4 ± 7.0%), while 228 animal 1 had the lowest values for minor abnormalities. No differences in minor abnormalities 229 values were detected among the three males. The most frequent abnormalities were simple bent 230 tail (Fig. 1F), which were recorded in 14.1 ± 15.3% of the samples, and bowed midpiece, which 231 were recorded in 4.6 ± 7.7% of the samples (Fig. 1E and 2C). Other abnormalities were detected 232 during the analysis, and are shown in Figure 2, including a narrow part at the base of the head, 233 proximal droplet (Fig. 1B), short and broad head (Fig. 1C), detached acrosome (Fig. 1D), 234 broken tail (Fig. 1F), strongly coiled tail (Fig. 1G), and detached normal head (Fig. 1H). 235 236 3.2. Morphometric evaluation 237 238 The total head length was 8.5 ± 0.5 µm, total head width was 4.4 ± 1.0 µm, total 239 midpiece length was 11.5 ± 2.0 µm, total tail length was 41.3 ± 1.7 µm, and total length was 240 61.2 ± 2.6 µm. Animal 1 had higher head length and width than the other two males. No 241 additional differences were observed among the animals. All results on morphometrics are 242 presented in Table 5. 243 244 3.3. Ultrastructural evaluation using scanning and transmission electron microscopy 245 246 SEM images were used to analyze the totality of the sperm analyzed, discerning head, 247 midpiece, and principal piece (tail). Electron microphotographs showed that the sperm had a 248 flattened head, dorso-ventrally, and, when the broad flat side was viewed, the head appeared 249 paddle-shaped, with a more squared base and a notable apical ridge being present along the 250 anterior side. The serrated band and the equatorial subsegment were rarely observed in cells, 251 but were detected more often than the equatorial segment. The midpiece and principal piece 42
252 had a smooth surface, divided by the annulus, and differed in thickness, as the midpiece was 253 slightly thicker than the principal piece (Fig. 2A-B). 254 Several abnormalities were visualized in S.E.M. images of the head. The narrow at base 255 head was evident as a drop-shaped head (Fig. 2C). Narrower and larger heads were notable due 256 to the discrepancy when compared to a normal one, but some retained the paddle-shape format 257 (Fig. 2D). The abnormal contour was characterized as a completely different shaped-head that 258 had lost the paddle-shape pattern (Fig. 2E). The pyriform head was a pear-shaped structure that 259 was broader at the anterior region and narrower at the posterior region (Fig. 2F). Cytoplasmic 260 droplets were also observed as larger round areas of cytoplasmic buildup proximally or distally 261 in the midpiece (Fig. 2G). 262 The images obtained using TEM showed that the head contained the nucleus, enveloped 263 by the nuclear membrane, and was surrounded by the acrosome and the sub-acrosomal space at 264 the apex. The nucleus contained most of the head (Fig. 3A), and was electron-dense with 265 electron-lucent points. The acrosome was enclosed by inner and outer membranes, and 266 extended beyond the apex of the head to the equatorial segment, where it narrowed caudally. 267 The sub-acrosomal space was present in the sperm apex (Fig. 3B). The caudal portion of the 268 nucleus was surrounded with a post-acrosomal sheath (Fig. 3C). 269 The implantation fossa limited the nuclear posterior end, and led to the connecting 270 piece, which was formed of the capitulum, the proximal centriole, and segmented columns, 271 which were the structures mainly responsible for connecting the head to the tail (Fig. 3D). The 272 segmented columns ended as outer dense fibers appeared in the midpiece anterior region. 273 Mitochondria were observed close to outer dense fibers, forming a dense spiral. In longitudinal 274 view, it was counted 60 mitochondrial spirals. The annulus was present in the distal portion of 275 the midpiece, where the principal piece began (Fig. 3E-F). The axoneme was enclosed by outer 276 dense fibers, and was characterized by nine doublets of microtubules forming a cylindrical 277 bundle arranged around a pair of microtubules that continued until the end of the principal piece 278 (Fig. 3F). 279 The principal piece was composed of the axoneme and the outer dense fibers that were 280 encircled with a fibrous sheath and the plasma membrane (Fig. 3G). Fibers 1,5 and 6 were larger 281 than the others. The fibrous sheath was an electron-dense material attached to a central pair of 282 microtubules in fibers 3 and 8 (Fig. 3F). 283 Degenerated sperm were observed in the TEM, and were characterized by the presence 284 of a broken plasma membrane, cytoplasmic residue, acrosome degeneration, plasma membrane 285 extensions, and sub-membranous swelling (Fig. 4A-C). Also, strongly coiled tail was observed 43
286 with many middle and principal piece cross-sectional sections enclosed by the same membrane 287 and a disarranged mitochondria sheath and axoneme fibers (Fig. 4D-E). 288 289 4. DISCUSSION 290 291 Literature on semen analysis in gray brocket deer is scarce (Cursino & Duarte, 2016; 292 Duarte & Garcia, 1995; Rola et al., 2012). Thus, this study advances current knowledge on 293 certain aspects of the male reproductive biology of this species. Specifically, this study is the 294 first to analyze membrane integrity using HOST, mitochondrial activity, and ultrastructure 295 using SEM and TEM. 296 Electroejaculation is a widely used method for collecting semen from deer species 297 (Abreu, Martinez, Moraes, Juvenal, & Moreira, 2009; Monfort, Asher, et al., 1993; 298 Panyaboriban et al., 2016; Rola et al., 2013). In the present study, the chosen protocol combined 299 with anesthetic provided enough amounts (298.1 ± 120.5 µL) of high-quality sperm, without 300 any urine contamination. The volume recorded in this study reflected that obtained previously 301 for M. gouazoubira, collected using electroejaculation (Cursino & Duarte, 2016; Duarte & 302 Garcia, 1995). Other semen collection techniques have been used on M. gouazoubira, including 303 an adapted artificial vagina and female mannequin (Rola et al., 2012); however, a period of 304 conditioning is required, with insufficient semen volume being generated. In comparison, the 305 modified artificial vagina was used successfully for Cervus elaphus, with good quality semen 306 samples being obtained (Gizejewski, 2004). Also, post-mortem collection, directly from the 307 epididymis, has provided promising results in C. elaphus hispanicus (Martínez et al., 2008). 308 The pH values obtained (6.7 ± 0.5) in the current study, supported those obtained for other deer 309 species, including M. americana (Rola et al., 2013), Ozotoceros bezoarticus (Beracochea et al., 310 2014), Dama dama mesopotamica (Ekrami, Tamadon, Jahromi, Moghadas, & Seno, 2016), C. 311 elaphus (Gizejewski, 2004) and Elaphodus cephalophus (Panyaboriban et al., 2016). 312 High percentages of total and progressive motility, vigor, and mass movement were 313 recorded in the current study, and were slightly better than other species. The average 314 percentage of total motility of fresh semen samples was at least 80% in C. eldi thamin (Monfort 315 et al., 1993) and in C. elaphus during the mating season (Gizejewski, 2004). For fresh sperm 316 from O. bezoarticus, high total and progressive motility was also recorded (Beracochea et al., 317 2014). For M. nana and M. americana, mean values for motility and vigor were around 70% 318 and 3, respectively, in fresh semen samples (Abreu et al., 2009; Luciana Diniz Rola et al., 2013). 319 The mean mass movement for M. americana and Cervus unicolor was around 3 and 4, 44
320 respectively (Chacur et al., 2014; Rola et al., 2013). Thus, the sperm of gray brocket deer sperm 321 has good antioxidant ability (Darr et al., 2016), mitochondrial integrity (Paoli et al., 2011) and 322 effective osmolarity control capacity (Cooper & Yeung, 2003). Higher motility values might 323 be associated with higher conception rates (Guerra Neto, Chacur, Caldato, Silva, & Castilho, 324 2012). 325 The sperm concentrations documented in the present study were highly variable (94.9 ± 326 55.1 x 10⁷ sptz/mL). There are no reference values for gray brocket deer; however, mean sperm 327 concentrations ranging from 50.18 to 112.67 x 107 sperm/mL have been previously described 328 (Cursino & Duarte, 2016). The measurements obtained in the current study reflect these values. 329 Similar results were obtained for other cervids, including Dama dama, Odocoileus virginianus, 330 Elaphodus cephalophus, C. unicolor, and M. nana (Abreu et al., 2009; Chacur et al., 2014; 331 Fernández, Sestelo, Rivolta, & Córdoba, 2013; Kirschner & Rodenkirch, 2017; Panyaboriban 332 et al., 2016). 333 Compared to Iberian red deer and red brocket deer using fluorescent probes, gray 334 brocket deer exhibited higher viability (87.6 ± 4.5). Over 70% viability (average percentage of 335 intact membrane) was obtained from both epidydimal (Martínez et al., 2008) and ejaculation 336 fresh semen samples (Favoretto, Zanetti, & Duarte, 2012). Sperm viability analysis helped to 337 inform many studies investigating reproduction, as data on sperm survival and membrane 338 damage allows the best samples to be selected for cryopreservation (Favoretto et al., 2012; 339 Martínez et al., 2008). Thus, the samples obtained in the present study likely had high fertility 340 potential, based on the high percentage of live sperm (Anzar, He, Buhr, Kroetsch, & Pauls, 341 2005). 342 In this study, animal 1 (46.7 ± 0.3) had fewer viable cells than animal 2 (78.3 ± 8.5) and 343 3 (86.2 ± 10.6) when using HOST. This difference was caused by the higher number of 344 abnormal tail sperm counted during the morphological analysis of male 1, which directly 345 interfered with the results. The total mean percentage of viable cells recorded in this study (73.4 346 ± 18.3) was lower than that recorded for O. bezoarticus (85% to 96%) (Beracochea et al., 2014). 347 For thawed cryopreserved samples of C. elaphus hispanicus and in D. dama, the mean 348 percentage of viable cells was 27% (Martínez-Pastor et al., 2006) and 82.37% (Fernández et 349 al., 2013), respectively. Non-reactive cells on HOST might have suffered problems during the 350 maturation phase, developing a less effective capacity to control volume. This issue is important 351 to enhance fertility, because sperm are deposited in the uterus, which is a hypotonic medium, 352 and must maintain their hydrodynamic shape (Cooper & Yeung, 2003; Yeung & Cooper, 2002). 45
353 Thus, in general, the sperm of gray brocket deer had a healthy plasma membrane that could 354 maintain osmolarity inside the cell. 355 Mitochondrial activity is potentially correlated with the kinetic parameters of sperm 356 (Paoli et al., 2011). Despite animals 2 and 3 having higher levels of DAB 3 mitochondrial 357 activity in this study, lower motility and vigor values were not perceived. Thus, DAB 1 and 2 358 might be more strongly associated with motility and vigor parameters, because all three study 359 animals had good energetic levels, with most cells containing more than 50% active 360 mitochondria. In addition, motility appears to be independent, to a certain degree, from 361 mitochondrial activity in the semen of gray brocket deer, because the motility percentage was 362 higher than the percentage of spermatozoa with high mitochondrial activity. This phenomenon 363 was confirmed in the sperm of red deer sperm (Martínez-Pastor et al., 2008). Thus, further 364 studies are required on the semen of M. gouazoubira to confirm this finding. Previous studies 365 using a different method (fluorescence) also obtained similar results with, approximately 44% 366 to 66% active mitochondria in Iberian red deer (F. Martínez-Pastor et al., 2009) and 27% to 367 67% in red deer (Anel-López et al., 2015). Mitochondria generate energy in spermatozoa and 368 regulate cell death (Peña et al., 2009). Mitochondria also have an important role in sperm 369 capacitation, because 55S ribosomes conduct nuclear-encoded protein translation (Gur & 370 Breitbart, 2006). Therefore, mitochondria are a valuable indicator of the fertility potential of 371 semen. 372 Sperm morphometry is a component of morphological analysis that is used to evaluate 373 changes to the size of a cellular region, contributing towards determining a standard for sperm 374 size of different species (Beracochea et al., 2014). Species-specific morphometric description 375 could be used to perform computer-assisted sperm analysis of kinetic parameters and 376 morphology (Kirschner & Rodenkirch, 2017; Yániz, Soler, & Santolaria, 2015). Measurements 377 of M. gouazoubira sperm in the current study were similar to those described previously for 378 this species, although tail and total length had slightly lower means in the present work. The 379 head length and width of pampas deer (Beracochea et al., 2014), Iberian red deer (Soler et al., 380 2005), and Sambar deer (Mahre et al., 2014) were also similar to those of gray brocket deer 381 (head width: 4.4 ± 1.1 µm). 382 The morphometric evaluation of sperm in the present study differed to previous studies 383 in C. elaphus hispanicus (Soler et al., 2005), and in other mammals, including bulls, rams, and 384 stallions (Morrow & Gage, 2001). These difference might result in different cryopreserved 385 efficiency of semen from males of different species (Soler et al., 2005) and different fertility 386 potential among males (Hirai et al., 2001; Ward, 1998). Individual differences are hypothesized 46
387 to be caused by sperm competition (Cursino & Duarte, 2016; Ward, 1998). For boars, the group 388 with lower fertility had narrower and longer heads (Hirai et al., 2001). However, this 389 interpretation is subject to controversy (Gomendio, Tourmente, & Roldan, 2011; Santolaria, 390 Vicente-fiel, Palacín, Fantova, & Blasco, 2015), requiring further confirmation for 391 Artiodactyls. For passerine birds, increased midpiece length is positively correlated to ATP 392 concentrations; however, ATP concentrations are not correlated to sperm velocity (Rowe, 393 Laskemoen, Johnsen, & Lifjeld, 2013). In comparison, the sperm of mice with longer midpiece 394 had faster velocity (Firman & Simmons, 2010). However, further studies are necessary to 395 confirm this phenomenon for gray brocket deer. In general, discrete differences in 396 morphometric parameters might not influence the hydrodynamics of sperm and, so, would not 397 influence fertility (Beletti, Costa, & Viana, 2005). 398 The current study used Blom’s classification for morphological analysis (Blom, 1972), 399 because it is widely used for several species (Azerêdo, Esper, & Resende, 2001; Chacur et al., 400 2014; Enciso et al., 2011; Gizejewski, 2004; Tsuneda et al., 2015). This classification also 401 provides the proportion of major and minor defects, with the latter being the least deleterious 402 to fertility (Blom, 1972). Studies investigating how major and minor defects influence the 403 fertility of sperm remain limited in most species. However, in bovine, major and total defects 404 were significant for predicting conception rates (Oliveira et al., 2013). Thus, studies are 405 required to confirm the assumptions of this classification system. 406 The current study obtained high values for normal morphology, with animal 3 showing 407 the highest average of normal sperm (85.4 ± 7.0 %). Normal morphology depends on factors 408 that occurred in the final spermatogenic cycle, and have an influence on spermatogenesis, sperm 409 transport, and sperm maturation on the epididymis (Brito, 2007). Different management 410 systems and diets can also affect sperm morphology (Ramachandran & Singh, 2017). 411 The number of major abnormalities and minor abnormalities obtained in the current 412 study was similar to that obtained for other deer species. The mean major and minor 413 abnormalities in the sperm of M. americana were approximately 8% and 18%, respectively 414 (Rola et al., 2013). For C. unicolor, individual differences were observed, with major 415 abnormalities ranging from 3% to 19% and minor abnormalities ranging from 3% to 46% 416 (Chacur et al., 2014). 417 Moreover, a simple bent tail was the most frequent pathology documented in the sperm 418 of gray brocket deer, especially in animal 1. The total mean obtained for gray brocket deer was 419 greater than that obtained for M. nana (1.5%) (Abreu et al., 2009) and M. americana (1.87%) 420 (Rola et al., 2013). Coiled tails indicate that spermatogenesis disturb has been disturbed, and 47
421 could be caused by elevated testis temperature in bulls (Barth & Bowman, 1994). Higher 422 ambient temperatures might be responsible for this phenomenon in our study, due to the tropical 423 nature of the study location (Fortaleza and Caucaia, Brazil). 424 Bowed and bent midpieces were the second most frequent abnormalities observed in 425 this study. The frequency of these abnormalities was similar to that detected in E. cephalophus 426 (3.7 to 4.8%) (Panyaboriban et al., 2016) and was lower than that detected in O. bezoarticus 427 (11.15%) (Beracochea et al., 2014) and M. nana (7%) (Abreu et al., 2009). Midpiece defects 428 might be positively correlated with apoptosis and negatively correlated with mitochondrial 429 membrane potential (Aziz, Said, Paasch, & Agarwal, 2007; Love, 2011). Thus, the frequency 430 of bowed midpieces (10.1 ± 11.4 %) in animal 2 might have partially influenced mitochondrial 431 activity. In addition, selenium deficiency causes midpiece abnormalities and mitochondria 432 damage (Li guang et al., 2010). 433 The SEM analysis confirmed the presence of the paddle-shaped head of sperm from 434 gray brocket deer, which was similar to that observed in other deer species. Also, an equatorial 435 segment is common in certain deer species, but is difficult to discern. Most likely, the 436 phylogenetic proximity is accountable for those similarities (Mahre et al., 2014; Meisner et al., 437 2005). The equatorial subsegment has been observed only in artiodactylans so far, whereas, its 438 implications are unclear (Meisner et al., 2005). 439 TEM also helps to identify normal and pathological sperm in greater details (Pesch et 440 al., 2006). It is possible to detect abnormalities in the acrosome, nucleus, plasma membrane, 441 mitochondria, and axoneme using TEM images (Ackerman, Reinecke, & Els, 1996; Bartoov et 442 al., 1980; Brito, Sertich, Stull, Rives, & Knobbe, 2010). The current study provided a complete 443 description of the spermatozoa structures in physiological and pathological situations. The 444 nucleus contained small, electron-lucent areas represented sub-condensed chromatin (Bartoov 445 et al., 1980). These areas are commonly observed in the sperm of other mammals, including 446 ring-tailed coati (Silva et al., 2015), six-banded armadillo (Sousa et al., 2013), jaguar (Silva et 447 al., 2019) and American black bear (Brito et al., 2010). 448 The mitochondrial sheath of gray brocket deer formed about 60 spirals, with this number 449 being similar to that documented in the jaguar with 54 spirals (Silva et al., 2019), the ring-tailed 450 coati with 55 spirals (Silva et al., 2015), the American black bear with 59 spirals (Brito et al., 451 2010), and stallions with 60 spirals (Brito, 2007). Interestingly, impala (bovid) sperm had 48 452 spirals (Ackerman et al., 1996). Additional studies are required to correlate this characteristic 453 to some sort of advantage to reproduction in these species. Furthermore, TEM could provide 454 important information for morphological studies (Bartoov et al., 1980), including how different 48
455 extenders influence cryopreservation (Badr, Rawash, Ghada, Assi, & Hasan, 2015; Khalil, El- 456 Harairy, Zeidan, & Hassan, 2019) and how diet affects the characteristics of semen (Li guang 457 et al., 2010; Younan, El-Gabry, & Morsy, 2017). 458 459 5. CONCLUSIONS 460 461 In conclusion, M. gouazoubira has similar semen traits to other deer species, as well as 462 some peculiarities regarding semen parameters and ultrastructural features. The data obtained 463 by this study provides baseline values of diverse semen parameters and descriptions that could 464 be used to detect disturbances on spermatogenesis, spermiogenesis, and sperm capacitation. In 465 addition, the information collected by the current study could aid cryopreservation protocols, 466 the selection of viable males, and future germplasm research that could be used to conserve this 467 species and other deer species at risk of extinction. 468 469 ACKNOWLEDGEMENTS 470 The authors would like to thank the Aba-Yby Institute – Ecopoint Environmental Education 471 Ltd. and the Claro Commercial Wild Animal Breeding Farm for supplying the animals used in 472 the experiment; to the Carnivore Reproduction Laboratory of Ceara State University, the Small 473 Ruminant Semen Technology Laboratory of Ceara State University, the Compared 474 Experimental Morphology Laboratory of Ceara State University, and the Central Analítica - 475 UFC/CT - INFRA/MCTI - SISANO/Pró-Equipamentos CAPES for the technical support. 476 We would also like to thank these funding agencies for financial support: FUNCAP (Fundação 477 Cearense de Apoio ao Desenvolvimento Científico e Tecnológico – Ceara’s Foundation of 478 Scientific and Technologic Development Support), CNPq (Conselho Nacional de 479 Desenvolvimento Científico e Tecnológico - National Council for Scientific and Technological 480 Development), CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - 481 Brazilian Federal Agency for the Support and Evaluation of Graduate Education) through 482 research grants (CAPES/Cofecub number 88881.142966/2017-01). 483 484 CONFLICTS OF INTEREST 485 The authors have no competing interests to declare. 486 487 AUTHORS CONTRIBUTIONS 49
488 DIAT was the counselor; DIAT, MBS, DMSC, and BFB designed the methodology; DMSC, 489 MBS, BFB, HVRS performed the experiment; LRR performed handling and anaesthesia; 490 FAFJR and JSAME helped with electron microscopic and morphometric analysis; DIAT and 491 JSAME provided equipment and reagents; DMSC drafted the manuscript and DIAT, LMCP, 492 MBS, HVRS and BFB revised it critically.
493 494 ORCID 495 Dárcio Ítalo Alves Teixeira
496 http://orcid.org/0000-0003-1799-1675
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692 motility. Fertility and Sterility, 95(7), 2315–2319. 693 https://doi.org/10.1016/j.fertnstert.2011.03.059 694 Peña, F. J., Rodríguez Martínez, H., Tapia, J. A., Ortega Ferrusola, C., González Fernández, 695 L., & MacÍas García, B. (2009). Mitochondria in mammalian sperm physiology and 696 pathology: A review. Reproduction in Domestic Animals, 44(2), 345–349. 697 https://doi.org/10.1111/j.1439-0531.2008.01211.x 698 Pesch, S., Bostedt, H., Failing, K., & Bergmann, M. (2006). Advanced fertility diagnosis in 699 stallion semen using transmission electron microscopy. Animal Reproduction Science, 700 91(3–4), 285–298. https://doi.org/10.1016/j.anireprosci.2005.04.004 701 Ramachandran, N., & Singh, N. P. (2017). Effect of management systems and seasons on 702 sperm abnormalities in Jamunapari bucks semen. Indian Journal of Animal Research, 703 51(6), 1138–1143. https://doi.org/10.18805/ijar.v0iof.7808 704 Rola, L. D., Zanetti, E. S., & Duarte, J. M. B. (2012). Avaliação de dois métodos para 705 condicionamento e coleta de sêmen em quatro espécies do gênero Mazama. Pesquisa 706 Veterinaria Brasileira, 32(7), 658–662. https://doi.org/10.1590/S0100- 707 736X2012000700013 708 Rola, L.D., Zanetti, E. D. S., & Duarte, J. M. B. (2013). Evaluation of semen characteristics 709 of the species Mazama americana in captivity. Animal Production Science, 53(5), 472– 710 477. https://doi.org/10.1071/AN12247 711 Rowe, M., Laskemoen, T., Johnsen, A., & Lifjeld, J. T. (2013). Evolution of sperm structure 712 and energetics in passerine birds. Proceedings of the Royal Society B: Biological 713 Sciences, 280(1753). https://doi.org/10.1098/rspb.2012.2616 714 Santolaria, P., Vicente-fiel, S., Palacín, I., Fantova, E., & Blasco, M. E. (2015). Predictive 715 capacity of sperm quality parameters and sperm subpopulations on field fertility after 716 artificial insemination in sheep. Animal Reproduction Science, 163, 82–88. 717 https://doi.org/10.1016/j.anireprosci.2015.10.001 718 Silva, H. V.R., Magalhães, F. F., Ribeiro, L. R., Souza, A. L. P., Freitas, C. I. A., de Oliveira, 719 M. F., … Silva, L. D. M. (2015). Morphometry, morphology and ultrastructure of ring- 720 tailed coati sperm (Nasua nasua Linnaeus, 1766). Reproduction in Domestic Animals, 721 50(6), 945–951. https://doi.org/10.1111/rda.12613 722 Silva, Herlon Victor Rodrigues, Nunes, T. G. P., Ribeiro, L. R., Freitas, L. A. de, de Oliveira, 723 M. F., Assis Neto, A. C. de, … Silva, L. D. M. da. (2019). Morphology, morphometry, 724 ultrastructure, and mitochondrial activity of jaguar (Panthera onca) sperm. Animal 725 Reproduction Science, 203(October 2018), 84–93. 56
726 https://doi.org/10.1016/j.anireprosci.2019.02.011 727 Soler, C., Gadea, B., Soler, A. J., Fernández-Santos, M. R., Esteso, M. C., Núñez, J., … 728 Garde, J. J. (2005). Comparison of three different staining methods for the assessment of 729 epididymal red deer sperm morphometry by computerized analysis with ISAS®. 730 Theriogenology, 64(5), 1236–1243. https://doi.org/10.1016/j.theriogenology.2005.02.018 731 Sousa, P. C., Santos, E. A. A., Bezerra, J. A. B., Lima, G. L., Castelo, T. S., Fontenele-Neto, 732 J. D., & Silva, A. R. (2013). Morphology, morphometry and ultrastructure of captive six- 733 banded armadillo (Euphractus sexcinctus) sperm. Animal Reproduction Science, 140(3– 734 4), 279–285. https://doi.org/10.1016/j.anireprosci.2013.05.015 735 Taber, A., Novaro, A., Neris, N., & Colman, F. (1997). The food habits of sympatric jaguar 736 and puma in the Paraguayan Chaco. Biotropica, 29(2), 204–213. 737 Tsuneda, P. P., Duarte, M. F., Silva, L. E. S. e, Jorge, A. A., Hatamoto-Zervoudakis, L. K., & 738 Paz, R. C. R. da. (2015). Análise seminal e padronização da coloração eosina-nigrosina 739 em tamanduá-bandeira (Myrmecophaga tridactyla). Revista Brasileira de Ciência 740 Veterinária, 22(3–4), 198–201. https://doi.org/10.4322/rbcv.2016.014 741 Ward, P. I. (1998). Intraspecific variation in sperm size characters. Heredity, 80(6), 655–659. 742 https://doi.org/10.1046/j.1365-2540.1998.00401.x 743 Yániz, J. L., Soler, C., & Santolaria, P. (2015). Computer assisted sperm morphometry in 744 mammals: A review. Animal Reproduction Science, 156, 1–12. 745 https://doi.org/10.1016/j.anireprosci.2015.03.002 746 Yeung, C., & Cooper, T. (2002). Effects of the ion-channel blocker quinine on human sperm 747 volume, kinematics and mucus penetration, and the involvement of potassium channels. 748 Molecular Human Reproduction, 7(9), 819–828. https://doi.org/10.1093/molehr/7.9.819 749 Younan, G. E., El-Gabry, H. E., & Morsy, W. A. (2017). Semen quality, testosterone level, 750 some enzymes activities and fertility of male apri rabbits treated with different levels of 751 olive leaf extract. Egyptian J. Nutrition and Foods, 20(2), 265–283. 752 753 754 755 756 757 758 759 57
760 Figure legends 761 Fig. 1. Photomicrography of gray brocket deer (Mazama gouazoubira) sperm on eosin-nigrosin smears at 1000X magnification. (A): Normal sperm 762 in morphological evaluation. (B): Narrow at base head with proximal droplet (arrow). (C): Short and broad head with proximal droplet (arrow). 763 (D) Detached acrosome and simple bent tail (arrow). (E): Bowed midpiece (arrow). (F): Broken tail (left) and simple bent tail (right). (G): Strongly 764 coiled tail (arrow). (H): Detached normal head. Scale bar = 10 µm.
765 58
766 Fig. 2. Electron microphotography of gray brocket deer (Mazama gouazoubira) sperm in scanning electron microscopy evaluation. (A): Normal 767 sperm in total visualization with regions identified as the head (H), midpiece (MP), annulus (AN) and principal piece (PP). Scale bar = 10 µm. (B): 768 Normal head presenting apical ridge (AR), equatorial subsegment (ES) and serrated band (SB). Scale bar = 3 µm (C): Narrow at base head and 769 bent midpiece. Scale bar = 10 µm. (D): Narrow (red arrow) and short broad heads (white arrow). Scale bar = 10 µm. (E): Abnormal head contour. 770 Scale bar = 3 µm (F): Pyriform head. Scale bar = 3 µm. (G): Distal droplet. Scale bar = 10 µm.
771 59
772 773 Fig. 3. Electron microphotography of normal gray brocket deer (Mazama gouazoubira) sperm structures in transmission electron microscopy 774 evaluation. AC: acrosome; AX: axoneme; CAP: capitulum; FS: Fibrous sheath; IAM: inner acrosomal membrane; IF: implantation fossa; MS: 775 mitochondrial sheath; NM: nuclear membrane; NU: nucleus; OAM: outer acrosomal membrane; ODF: outer dense fibers; PAS: post-acrosomal 776 sheath; PC: proximal centriole; PM: plasma membrane; SAS: sub-acrosomal space; SC: segmented columns; red arrows: electron-lucent areas. 777 (A): Sperm head and midpiece. (B): Sperm head in apical region. (C): Post-acrosomal region of the head. (D): Implantation fossa region. (E): 778 Distal midpiece region in longitudinal view. (F): Midpiece and principal piece in cross-sectional view. Numbers 1, 3, 5, 6, and 8 refer to ODF. (G): 779 Principal piece in longitudinal view.
780 60
781 Fig. 4. Electron microphotography of gray brocket deer (Mazama gouazoubira) sperm in transmission electron microscopy evaluation. (A-C): 782 Degenerated sperm. BPM: broken plasma membrane; CR: cytoplasmic residue; DAC: degeneration of acrosome; E: plasma membrane extensions; 783 PM: plasma membrane; S: sub-membranous swelling. (D-E): Strongly coiled tail.
784 61
Table 1 City, age, weight, proved fertility, housing with female and origin about the three gray brocket deer (Mazama gouazoubira).
Male Location Age Weight (kg) Proved Housed with Origin/born (city, state) (approximately) fertility female Male 1 Fortaleza, CE 3 years 14 No No Captive
Male 2 Caucaia, CE 5 years 15 Yes Yes Captive
Male 3 Fortaleza, CE 3 years 15,6 No Yes Captive
Table 2 General seminal parameters for gray brocket deer (Mazama gouazoubira). Values (Mean ± SD), (n = 8 ejaculates). Total Progressive Mass Concentration Viability Membrane Male pH Volume (µL) motility Vigor motility (%) movement (x 10⁷ sptz/mL) (%) integrity (%) (%) 1 7 ± 0 a 170 ± 28.3 a 97 ± 2.8 a 87.5 ± 10.6 a 4.7 ± 0.3 a 4.5 ± 0.7 a 48 ± 16.3 a 85.5 ± 4.2 a 46.7 ± 0.3 a 2 6.7 ± 0.6 a 365 ± 123.8 a 97.7 ± 2.3 a 86.7 ± 5.8 a 4.8 ± 0.3 a 4.3 ± 0.6 a 77 ± 40.4 a 85.2 ± 5.0 a 78.3 ± 8.5 b 3 6.5 ± 0.5 a 316 ± 106.9 a 95.7 ± 4.9 a 86.7 ± 5.8 a 4.8 ± 0.3 a 4.3 ± 0.6 a 144.2 ± 50.4 a 91.3 ± 1.4 a 86.2 ± 10.6 b Total 6.7 ± 0.5 298.1 ± 120.5 96.7 ± 3.2 86.9 ± 5.9 4.8 ± 0.2 4.4 ± 0.5 94.9 ± 55.1 a 87.6 ± 4.5 73.4 ± 18.3 Different letters show significant difference among males (P < 0.05).
62
Table 3 Evaluation of mitochondrial activities in gray brocket deer sperm (Mazama gouazoubira). Values (Mean ± SD), (n = 8 ejaculates).
Male DAB 1 (%) DAB 2 (%) DAB 3 (%) DAB 4 (%) 1 74.5 ± 6.4 a 20.7 ± 6.7 a 3.75 ± 0.3 a 1 ± 0.7 a 2 32.8 ± 10.9 a 42.7 ± 9.9 a 21.8 ± 2.5 b 2.7 ± 2.2 a
3 39.3 ± 23.6 a 37.8 ± 17.5 a 19.2 ± 13.2 b 3.7 ± 5.5 a Total 45.7 ± 22.9 35.4 ± 14.4 16.3 ± 10.7 2.6 ± 3.4 Different letters show significant difference among males (P < 0.05).
63
Table 4 Morphological evaluation in gray brocket deer sperm (Mazama gouazoubira). Values (Mean ± SD), (n = 8 ejaculates).
1 2 3 Total
b b 85.4 ± 7.0 Normal sperm 53.7 ± 6.0 72.5 ± 10.2 a 72.7 ± 14.8 Major abnormalities 5 ± 2.1 a 17.7 ± 11.0 a 4.0 ± 2.6 a 9.4 ± 9.2 Knobbed sperm 0.2 ± 0.3 0.2 ± 0.3 0 ± 0 0.1 ± 0.2 Diadem defect (pouch 0.2 ± 0.3 0.3 ± 0.6 0.3 ± 0.6 0.3 ± 0.5 formation) Pyriform head 0 ± 0 0.3 ± 0.3 0.7 ± 1.1 0.4 ± 0.7 Narrow at base head 0 ± 0 0.3 ± 0.3 0.8 ± 1.0 0.4 ± 0.7 Abnormal head contour 0 ± 0 0 ± 0 0.2 ± 0.3 0.1 ± 0.2 Small abnormal heads 0.2 ± 0.3 0.3 ± 0.3 0 ± 0 0.2 ± 0.3 Detached pathological 0 ± 0 0.3 ± 0.6 0 ± 0 0.1 ± 0.4 heads Bowed midpiece 1.5 ± 0.7 10.1 ± 11.4 1.2 ± 1.2 4.6 ± 7.7 Proximal droplets 0 ± 0 0.2 ± 0.3 0 ± 0 0.1 ± 0.2 Stump tail 0.5 ± 0.7 0 ± 0 0 ± 0 0.1 ± 0.4 Strongly coiled or folded 2.2 ± 1.0 5.5 ± 3.6 0.8 ± 0.3 2.9 ± 2.9 tail (dag defect) Teratoid forms 0 ± 0 0.2 ± 0.3 0 ± 0 0.1 ± 0.2 a b 10.7 ± 9.3 Minor abnormalities 41.2 ± 8.1 9.8 ± 3.7 b 18 ± 15.6 Detached acrossome 0 ± 0 0.2 ± 0.3 0.3 ± 0.3 0.2 ± 0.3 membranes Narrow heads 0.2 ± 0.3 0.3 ± 0.3 0 ± 0 0.2 ± 0.3 Small normal heads/ Giant and short broad 1.5 ± 0 0.5 ± 0.5 0.5 ± 0 0.7 ± 0.5 heads Detached normal heads 0.7 ± 0.3 3.0 ± 2.6 0.2 ± 0.3 1.4 ± 2.0 Abaxial implantation 0.5 ± 0.7 1.0 ± 1.3 0.2 ± 0.3 0.6 ± 0.9 Distal droplets 0 ± 0 0.2 ± 0.3 0 ± 0 0.1 ± 0.2 Simple bent tail 36.5 ± 7.0 3.6 ± 1.4 9.5 ± 9.8 14.1 ± 15.3 Broken tail 1.5 ± 0.0 0.2 ± 0.3 0 ± 0 0.4 ± 0.7 Terminally coiled tail 0.2 ± 0.3 0.8 ± 1.4 0 ± 0 0.4 ± 0.9 Different letters show significant difference among males (P < 0.05).
64
Table 5 Sperm morphometrical evaluation in three gray brocket deer (Mazama gouazoubira). Values
Head length Head width Midpiece Tail length Total length Male (µm) (µm) length (µm) (µm) (µm) 1 8.9 ± 0.5 a 4.7 ± 0.3 a 11.7 ± 0.7 a 40.4 ± 1.5 a 61.0 ± 1.8a 2 8.3 ± 0.4 b 4.3 ± 0.3 b 11.3 ± 3.1 a 41.5 ± 1.6 a 61.1 ± 3.5a 3 8.3 ± 0.4 b 4.2 ± 1.5 b 11.7 ± 0.5 a 41.6 ± 1.7 a 61.5 ± 1.8a Total 8.5 ± 0.5 4.4 ± 1.0 11.5 ± 2.0 41.3 ± 1.7 61.2 ± 2.6 (Mean ± SD), (n = 8 ejaculates). Different letters show significant difference among males (P < 0.05).
65
7 CAPÍTULO 2
Caracterização morfológica e ultrassonográfica testicular de veados-catingueiros (Mazama gouazoubira, Fischer, 1814) em diferentes estados reprodutivos.
Testicular morphological and ultrasonographic characterization of male gray brocket deers (Mazama gouazoubira, Fischer, 1814) in different reproductive status.
Duanny M. de S. Cunha, Mírley Barbosa de Souza, Bruna Farias Brito, Vítor Lima Torres, Thalles Gothardo Pereira Nunes, Samuel Salgado Tavares, Daniel de Araujo Viana, Lúcia Daniel Machado da Silva, Dárcio Ítalo Alves Teixeira
Periódico: Reproduction in Domestic Animals (a ser submetido) Qualis A2 66
1 Testicular morphological and ultrasonographic characterization of male gray brocket 2 deers (Mazama gouazoubira, Fischer, 1814) in different reproductive status.
3 Gray brocket deer testes morphological and sonographic characteristics
4
5 Duanny Murinelly de Souza Cunha1, Mírley Barbosa de Souza1, Bruna Farias Brito1, Vítor 6 Lima Torres1, Thalles Gothardo Pereira Nunes1, Samuel Salgado Tavares2, Daniel de Araujo 7 Viana3, Lúcia Daniel Machado da Silva1, Dárcio Ítalo Alves Teixeira1*
8 1 Faculty of Veterinary Medicine, Ceara State University, Fortaleza, Brazil.
9 2 Aba-Yby Institute – Ecopoint Environmental Education Ltd., Fortaleza, Brazil.
10 3 Pathovet Laboratory, Fortaleza, Brazil.
11 *Corresponding author: Dárcio Ítalo Alves Teixeira 12 E-mail address: [email protected] 13 Phone: +55 85 31019850
14
15 Summary
16 Gray brocket deer (Mazama gouazoubira, Fischer, 1814) populations have been declining due 17 to human intervention. Yet, only a few studies have assessed sonographic testicular 18 characteristics in cervids. Considering the relevance of monitoring testicular size, blood flow, 19 and parenchyma, the present study aims to establish baseline information on scrotal 20 circumference, testicular volume, and spectral Doppler parameters, to describe differences 21 amongst adult male gray brocket deer in different reproductive status, and to correlate 22 ultrasound parameters with testes size measurements. Six adult male gray brocket deers were 23 used in the study. Scrotal circumference and testicular volume were measured. B mode 24 ultrasound images of testes (longitudinal and cross-sectional views) and epididymes were 25 subjected to computer-assisted analysis, obtaining the numerical pixel values (NPV) and pixel 26 standard deviation (PSD). Using spectral Doppler, supratesticular artery blood flow velocities, 27 resistivity, and pulsatility indices were obtained. Semen was analyzed through total motility, 28 vigor, and concentration tests. Three animals were normospermic (F+) and three were 29 oligo/azoospermic (F-). F+ group presented significantly higher scrotal circumference, 67
30 testicular volume, and testes cross-sectional NPV and PSD. Significant correlations were 31 observed between scrotal circumference and longitudinal (r = 0.76) and cross-sectional testes 32 NPV (r = 0.89), and testicular volume was correlated with longitudinal (r = 0.78) and cross- 33 sectional testes NPV (r = 0.91) and with cross-sectional testes PSD (ρ = 0.82). Thus, it is 34 suggested that scrotal circumference, testicular volume, and testes NPV are good indicators of 35 male reproductive health in gray brocket deer and may help with better male selection in the 36 species.
37 Keywords:
38 Wild animals, Cervidae, Andrology, Scrotal circumference, Pixel intensity, 39 Dopplervelocimetry.
40
41 1. INTRODUCTION
42 Commonly found in South America, gray brocket deers (Mazama gouazoubira) are 43 decreasing in number due to the extension of human populations. However, it is globally 44 classified as of “least concern” by the International Union for Conservation of Nature (Black- 45 Decima & Vogliotti, 2016). Furthermore, knowledge on male reproductive biology of this 46 species remains limited (Barrozo et al., 2001; Duarte & Garcia, 1995). Considering that 47 developing assisted reproduction programs is an important tool for species conservation (Pintus 48 & Ros-Santaella, 2014), improved diagnostic techniques may help with male selection, 49 increasing conservation programs efficiency (Ungerfeld, Villagrán, Lacuesta, Vazquez, & 50 Pérez, 2017).
51 Ultrasonography of testes, epididymis, and accessory glands has proven to be a valuable, 52 and non-invasive technique for the assessment of genital macroscopic morphology and 53 pathology (using B mode) and testicular blood flow (with Doppler technique) in several 54 mammalian species (Ali et al., 2011; Kutzler, Tyson, Grimes, & Timm, 2011; Ribeiro, Quirino, 55 Junior, & Pacheco, 2017; Samir, Nyametease, Nagaoka, & Watanabe, 2018). Yet, few studies 56 have investigated male deer reproductive system. Previous studies have assessed male 57 reproductive organs size, their sonographic appearance and testis pixel intensity of B mode 58 images (Goeritz et al., 2003; Ungerfeld et al., 2017). In domestic species, testes pixel intensity 59 has been used to objectively identify differences in testicular parenchyma during sexual 60 development (Bartlewski, Giffin, Oluwole, & Hahnel, 2017), after scrotal insulation (Arteaga, 68
61 Barth, & Brito, 2005) and to use it as a semen quality predictor (Ahmadi et al., 2012). Also, the 62 Doppler technique has aided in diagnosing blood flow changes in testicular artery in different 63 seasons (Hedia, El-Belely, Ismail, & El-Maaty, 2019) and in males with fertility disorders 64 (Kutzler et al., 2011; Ortiz-Rodriguez et al., 2017).
65 In addition, estimating testes size (scrotal circumference and testicular volume) is a 66 cheap, non-invasive method, widely used in several Cervidae species (Cheng et al., 2004; Costa 67 et al., 2011; Gosch, Bartolomaeus, & Fischer, 1989; Martinez-Pastor et al., 2005; Monfort et 68 al., 1993). Scrotal circumference is essential to breeding soundness examination in domestic 69 ruminants (Ahmadi et al., 2012; Barth and Waldner, 2002) and it has been associated with 70 fertility in rams (Duguma et al., 2002) and in bulls (Singh, Brar, & Cheema, 2014).
71 Therefore, considering the poor literature about ultrasound and testis size 72 characterization in gray brocket deer, the aims of this study were (1) to establish baseline 73 information on scrotal circumference, testicular volume, and spectral Doppler parameters for 74 the species, (2) to describe differences found between deer in different reproductive status, and 75 (3) to correlate ultrasound parameters with testes size measurements.
76
77 2. MATERIALS AND METHODS
78 2.1. Animals
79 The study was approved by Ceara State University Ethics Committee (number 7913746- 80 2017) and by the System of Authorization and Information on Biodiversity – SISBIO (number 81 60925).
82 Six adult male gray brocket deer (M. gouazoubira) were used in the study. The 83 evaluations were performed from January to December 2018. Two evaluations were completed 84 on each male, except on male 6, which was evaluated once (n = 11). The males were housed in 85 private institutions: two were at Aba-Yby Institute – Ecopoint Environmental Education Ltd. 86 (Fortaleza, CE, Brazil) and four were at Claro Commercial Wild Animal Breeding Farm 87 (Caucaia, CE, Brazil). The males were subjected to the usual management of each institution 88 above. At Claro Farm, the males were fed equine food, grass (Cybodon dactylon or Brachiaria 89 sp.) and roots (e.g. carrots and yuca) daily. At Aba-Yby Institute, males were offered ovine 90 food, fruits, and grass daily. At both institutions, the males received water ad libitum. More 91 information about the males is provided in Table 1. 69
92 2.2. Physical evaluation
93 Firstly, all males were sedated using 5 to 10 mg/kg ketamine hydrochloride (Cetamin, 94 Syntec, Brazil) and 0.5 to 1.5 mg/kg xylazine hydrochloride (Xilazina 10%, Venco, Brazil), 95 both injected intramuscularly (Cursino and Duarte, 2016; Duarte and Garcia, 1995). Once the 96 anesthesia took effect, a physical exam was performed. The testicular consistency was assessed 97 by palpation. Scrotal circumference (cm) was obtained using a measuring tape and testicular 98 volume (cm3) was obtained using a pachymeter and following the formula: 4/3 × π × ABC, 99 where A = width/2, B = height/2, C = length/2 (Costa et al., 2011).
100 2.3. Semen collection and general assessment 101 Males were lying in lateral decubitus position. The penis and prepuce were cleaned with 102 saline solution (sodium chloride 0.9%) and feces excess were removed from the rectum before 103 introducing the probe with the electrodes positioned towards the prostatic surface. Semen was 104 collected using an electroejaculator (Neovet Autoejac v2, Neovet, Uberaba, Brazil). The 105 procedure consisted of three sets of stimuli, with ten stimuli for each voltage: the first series 106 starting with 4 V, 5 V, and 6 V; the second with 5 V, 6 V, and 7 V; and the third with 6 V, 7 V, 107 and 8 V. Each series was separated by a 5 minute interval rest (Monfort et al., 1993). The semen 108 was collected in a 50 mL conical tube. 109 The microscopic parameters of total motility (0 – 100%) and vigor (score 0 to 5) were 110 assessed using a light microscope at 400X magnification. A 10 µL fresh semen aliquot was 111 added to a solution containing 2 mL 1 % formaldehyde-saline (0.9 % sodium chloride solution). 112 The concentration of sperm was quantified by counting the cells in five fields in a Neubauer 113 chamber with a light microscope at 400X magnification (Rola, Zanetti, & Duarte, 2013). Males 114 1, 2, and 4 were normospermic (high fertility potential; group F+). Male 3 appeared 115 oligospermic in the first collection, but azospermic in the second collection; and males 5 and 6 116 were azoospermic on both analyses (low or no fertility potential; group F-). 117 2.4.Ultrasound examination
118 After the physical exam, the ultrasound assessment was performed using Mindray 119 Z5Vet (Mindray®, Germany). Males 1 to 5 were evaluated twice using the B mode technique 120 (n = 10). Longitudinal and cross-sectional sections of testis and the epididymal tail were 121 evaluated with a convex transducer under the following parameters: 5 MHz, gain of 64, depth 122 of 16.6 (only on animal 1’s first evaluation, depth was 9.2), frame rate of 56, B dynamic range 123 of 110. Two images of each section were taken and saved. Afterwards, they were submitted to 70
124 a computerized analysis (spot-meter technique) using the software ImageJ (Wayne Rasband - 125 National Institute of Health, U.S.A.). Six 3 mm × 3 mm square regions of interest (ROI) in 126 testis longitudinal view, four in testis cross-sectional views, and two in epididymal images were 127 selected (Fig. 1). The mean pixel intensity (numerical pixel values, or NPV) and the pixel 128 heterogeneity (pixel standard deviation, or PSD) were calculated inside those ROI in each 129 image (Pozor et al., 2017). NPV were defined as gray-scale values of individual picture 130 elements ranging from 0 (absolute black) to 255 (absolute white).
131 Moreover, the spectral Doppler evaluation was performed on animals 1 to 6 (n = 11) 132 using a linear transducer at 10 MHz. The angle of insonation used was set at 0 º. At the region 133 of the spermatic cord, the supratesticular artery flow was detected with color Doppler, the gate 134 was positioned within the lumen of the vessel, and the equipment’s algorithm package was used 135 to calculate peak systolic velocity (PSV), end diastolic velocity (EDV), time-average maximum 136 velocity (TAMV), resistive index (RI) and pulsatility index (PI) during three waves of a cardiac 137 cycle (Ortiz-Rodriguez et al., 2017). The same operator performed all examinations.
138 2.5. Statistical analysis
139 The data was analyzed using the statistical software R-project© (The R Foundation, 140 Vienna, Austria), being submitted to the Cramer-Von Mises normality test and the Box-Cox 141 homoscedasticity test. For comparison of means between groups F+ and F-, the homogenous 142 and normally distributed parameters (PSV, EDV, TAMAX, RI, NPVs of testes longitudinal and 143 cross-sectional views, NPVs and PSDs of epididymis, PSDs of testes cross-sectional view, 144 scrotal circumference, testicular volume) were compared using a one-way ANOVA followed 145 by Student-Newman-Keuls (SNK) test. Logarithmic (Ln) transformation was performed on 146 EDV, TAMAX, PSDs of testes cross-sectional view and epididymal PSDs. The non-parametric 147 data (PI, PSDs of testes longitudinal view) were compared using a Kruskal-Wallis test followed 148 by SNK test. 149 Ultrasound parameters were correlated to testes size parameters using Pearson’s 150 correlation for parametric variables (PSV, RI, NPVs of testes longitudinal and cross-sectional 151 views, epididymal NPVs, scrotal circumference, and testicular volume) and Spearman’s 152 correlation for non-parametric variables (PI, EDV, TAMAX, PSDs of testes longitudinal and 153 cross-sectional view, and epididymal PSDs). The results are expressed as mean ± SD. 154 Significance was set at P < 0.05. 155 71
156 3. RESULTS
157 3.1. Semen evaluation
158 Males 1, 2 and 4 ejaculated in both collections and presented good quality semen 159 (motility: 96.83 ± 3.71 %; vigor: 4.41± 0.38; concentration: 94.08 ± 65. 04 × 10⁷ sptz/ml). 160 Male 3 ejaculated a low count sperm (4 × 10⁷ sptz/ml) with poor motility (5 %) and vigor (1) 161 on its first collection (Table 2). On the second collection, it presented azoospermia. Males 5 162 and 6 were azoospermic in all collections.
163 Male 6 presented intense germ cell epithelial degeneration in the histopathological exam 164 which was compatible to testicular degeneration.
165 3.2. Physical examination
166 During physical examination, males 1, 2, 4, and 5 had normal consistence regarding 167 their testes, but soft ones were identified in males 3 and 6. F+ group showed wider scrotal 168 circumference (14.57 ± 1.19 cm) and larger testis volume (26.18 ± 4.94 cm3) when compared 169 to group F- (Table 3).
170 3.3. Ultrasound evaluation
171 During the subjective B mode evaluation, it was observed that testes and epididymis 172 were in the normal position, inside the scrotum. No abnormalities were noticed in the testicular 173 and epididymal parenchyma. Testicular parenchyma had a smooth echotexture. The 174 mediastinum was observed as a hyperechoic line (longitudinal view) or point (cross-sectional 175 view) in the center. The tunica albuginea was observed as a hyperechoic line surrounding the 176 parenchyma and enclosing it and, the scrotum skin, as thicker hyperechoic line. The epididymis 177 tail was slightly hypoechoic, comparing to testicular echogenicity, and it is also surrounded by 178 tunica albuginea and the skin (Fig. 2).
179 The waveform originated from supratesticular artery blood flow is shown in Figure 3. 180 As shown in Table 4, no significant differences were observed between the groups regarding 181 the spectral Doppler ultrasound parameters: PSV (F+: 10.66 ± 5.60 cm/s, F-: 9.77 ± 4.71 cm/s), 182 EDV (F+: 5.59 ± 2.17 cm/s, F-: 5.67 ± 2.13 cm/s), TAMAX (F+: 0.38 ± 2.62 cm/s, F-: 7.14 ± 183 2.77 cm/s), PI (F+: 0.89 ± 0.75, F-: 0.53 ± 0.12), and RI (F+: 0.40 ± 0.10, F-: 0.38 ± 0.06). 184 However, NPVs and PSDs of cross-sectional testes images were higher on F+ group (NPV: 185 69.88 ± 24.00, PSD: 10.78 ± 3.42) than F- group (NPV: 28.26 ± 13.75, PSD: 6.70 ± 1.84). 186 Longitudinal testis (F+: 66.16 ± 30.66, F-: 29.42 ± 12.42) and epididymal images NPVs (F+: 72
187 42.28 ± 14.32, F-: 24.95 ± 6.78) were not significantly different between the groups. Likewise, 188 longitudinal testes (F+: 13.92 ± 10.47, F-: 7.71 ± 3.41) and epididymal images PSDs (F+: 12.74 189 ± 6.59, F-: 10.77 ± 8.31) showed no significant differences.
190 3.4. Correlations between ultrasound and testis size parameters
191 Scrotal circumference was strongly correlated to testicular volume (r = 0.88, P = 192 0.0003). There was a moderate correlation between longitudinal testes NPVs and testicular size 193 parameters such as scrotal circumference (r = 0.76, P = 0.01) and testicular volume (r = 0.78, 194 P = 0.008). Cross-sectional testes NPVs were strongly correlated to scrotal circumference (r = 195 0.89, P = 0.0006) and testicular volume (r = 0.91, P = 0.0003). Cross-sectional testes PSDs 196 were strongly correlated to testicular volume (ρ = 0.82, P = 0.004), while the other ultrasound 197 parameters showed no significant correlations with testicular size parameters, as displayed on 198 Table 5.
199
200 4. DISCUSSION
201 The present results give basal information on testes and epididymis sonographic 202 appearance, testes perfusion, and testicular size in healthy male gray brocket deers as well as 203 pathological changes associated with azoospermia. To the best of our knowledge, this study is 204 the first to report changes in testicular echogenicity, scrotal circumference, and testicular 205 volume in normospermic and oligo/azoospermic gray brocket deer adult males and to correlate 206 these variables.
207 Scrotal circumference is a simple repeatable method of measuring testicular size which 208 is strongly correlated with testicular weight (Palasz, Cates, Barth, & Mapletoft, 1994), semen 209 production (Dana, Tegegne, & Shenkoru, 2000; Ugwu, 2009) and with fertility (Palasz et al., 210 1994). Testicular volume is also a simple method of measuring testes and is also correlated to 211 scrotal circumference (Ugwu, 2009). It is positively correlated to sperm motility and 212 morphology and negatively correlated to immature germ cells (Condorelli, Calogero, & La 213 Vignera, 2013). In the present study, scrotal circumference and testicular volume measurements 214 were different between normospermic and azoospermic deers. The source of these deer’s poor 215 semen quality and decreased testes size is possibly testicular degeneration (Bousmaha & 216 Khoudja, 2012; Javed, Khan, & Naz, 2001) which can be caused by multiple factors such as 217 advanced age, heat, medication (nitrofurans and benzimidazoles), nutritional deficiencies, 218 stress, trauma or corticosteroid therapy (Foster, 2009). The cause for sperm disturbances was 73
219 confirmed only in male 6, which presented intense germ cell epithelial degeneration compatible 220 to testicular degeneration in the histopathological analysis.
221 Unlike what might happen in rams (Batissaco et al., 2013), testicular degeneration may 222 not cause noticeable alterations in deer’s testes parenchyma using ultrasound subjective 223 evaluation. Similarly, no ultrasound subjective alterations were noted in bulls after scrotal 224 insulation. However, lower mean pixel intensity was also observed in bulls’ testes after scrotal 225 insulation, suggesting the occurrence of reduced echogenicity during a degenerative process. 226 Thus, computerized analysis of B mode images may help providing detailed and objective 227 information about subtle changes and have an important role in reproductive assessment 228 (Arteaga et al., 2005).
229 Some factors can influence testicular pixel intensity (NPV), such as age (Brito et al., 230 2012; Pozor et al., 2017) and genotype. In bulls, there was an increase in testicular echogenicity 231 (testicular pixel intensity) during sexual development, which is associated with improved 232 testicular growth and cell population expansion (Brito et al., 2012). Also, testes NPV were 233 moderately and positively correlated with inner and outer seminiferous tubules diameter in bulls 234 (Evans et al., 1996), and moderate to strong positive correlations were observed between testes 235 NPV and seminiferous tubules area and lumen in stallions (M. Pozor et al., 2017). Thus, it is 236 suggested that the animals in group F- had compromised testicular development and 237 spermatogenesis due to their lower testicular NPV than the ones in group F+. The deer in F+ in 238 the present study presented good seminal concentration and higher values for scrotal 239 circumference and testicular volume. This can also be linked with observations of a previous 240 study in bulls in which pixel intensity showed moderate positive correlations to spermatids 241 percentages and testis weight (Evans et al., 1996), meaning that the higher the NPV, the more 242 spermatids are observed and the larger are testes (in a physiological setting).
243 The F- group had lower testicular volume, NPVs and PSDs in cross-sectional testicular 244 images, suggesting higher protein levels and lower lipid contents were present the parenchyma 245 of those animals and those components account for echogenicity and echotexture of the 246 testicular parenchyma (Ahmadi et al., 2013).
247 About pixel heterogeneity, group F+ presented more heterogeneous testes (in cross- 248 sectional view), so higher PSD is associated to a better spermatogenic function. In dogs, more 249 heterogenous testes were associated with higher sperm output (Moxon et al., 2015) and, in 250 stallions, scrotal testes from young males were more heterogenous than retained testes (Pozor 251 et al., 2017). In addition, testes PSD (obtained through ultrasound evaluation in direct contact 74
252 with tunica albuginea) was positively correlated to lumen area of seminiferous tubules in rams 253 (Giffin, Franks, Rodriguez-Sosa, Hahnel, & Bartlewski, 2009) and stallions (Pozor et al., 2017). 254 In animals with spermatogenesis imbalance, the cell population is reduced inside seminiferous 255 tubules (Bousmaha & Khoudja, 2012), and their area is decreased too (Pozor et al., 2017). 256 Hence, it is suggested that a decrease in variation between anechoic and echogenic parenchyma 257 is observed in those males due to decrease in density of seminiferous tubules and consequent 258 cell population reduction inside these tubules.
259 In this study, moderate to strong correlations were observed between testes NPV and 260 scrotal circumference. The latter can be moderately associated with testes cell population 261 parameters. In bulls, scrotal circumference was positively correlated to daily sperm production 262 (Palasz et al., 1994) and negatively correlated to the degree of germinal epithelial loss (Madrid 263 et al., 1988), suggesting that changes on seminiferous tubules can affect scrotal circumference 264 measurements and testes echogenicity and echotexture.
265 Scarce literature describes spectral Doppler parameters of testicular artery. RI and PI of 266 testicular artery have been described in domestic animals, such as rams (Batissaco et al., 2013), 267 stallions (Pozor & McDonnell, 2004) and dogs (Souza et al., 2015) and in wild animals, such 268 as llamas and alpacas (Kutzler et al., 2011). Discrepancies in RI and PI may be observed 269 amongst males, suggesting spectral Doppler indices are highly variable (Batissaco et al., 2013). 270 Those parameters can be associated with semen quality (Biagiotti, Cavallini, Modenini, Vitali, 271 & Gianaroli, 2002; Hedia et al., 2019). Thus, describing spectral Doppler parameters in gray 272 brocket deer may contribute to establishing reference values for healthy males and aiding in 273 diagnosing vascular disturbs involving testes (Gumbsch et al., 2002; Ortiz-Rodriguez et al., 274 2017) or evaluating if a pre-existing pathology is affecting testicular blood supply (Günzel- 275 Apel, Möhrke, & Nautrup, 2001; Ortiz-Rodriguez et al., 2017).
276 In this study, no differences in spectral Doppler parameters were observed between the 277 two groups. Similarly, in fertile and subfertile stallions, there was no difference among blood 278 flow parameters in the supratesticular artery (Ortiz-Rodriguez et al., 2017; Pozor & McDonnell, 279 2004). On the contrary, in dogs with and without testicular tumor, differences in PSV and 280 TAMAX were observed (Günzel-Apel et al., 2001) and, in fertile and infertile dogs, PSV and 281 EDV were different (Souza et al., 2015). In camelids, RI showed no difference between fertile 282 and infertile males, but PSV and EDV were higher for fertile males (Kutzler et al., 2011). These 283 different results may be due to different locations of the evaluated vessel, species and 284 techniques. It is known that many factors can influence vascular changes, including age (Pozor 75
285 & McDonnell, 2004), ambient temperature, seasonality (Samir et al., 2018; Strina et al., 2016), 286 pathological conditions (Souza et al., 2015; Ortiz-Rodriguez et al., 2017), and anaesthesia 287 (Baumgartner et al., 2010). Thus, they may have influenced the spectral Doppler parameter’s 288 variability in the supratesticular artery of gray brocket deer.
289 290 5. CONCLUSION 291 In conclusion, changes in testicular tissue in deer with different reproductive status were 292 detectable using testes NPV, scrotal circumference, and testicular volume measurements where 293 lower values were observed in deer with reproductive disruptions. Thus, these techniques can 294 be feasible procedures and good indicators of male reproductive health in gray brocket deer, 295 supporting in male selection for assisted reproduction programs. The correlations between 296 testicular size and testes NPV and PSD can be associated with germ cell population and 297 spermatogenesis; however, further studies are required to confirm this association in the 298 species. 299 300 ACKNOWLEDGEMENTS 301 The authors would like to thank the Aba-Yby Institute – Ecopoint Environmental Education 302 Ltd. and the Claro Commercial Wild Animal Breeding Farm for supplying the animals used in 303 the experiment; to the Carnivore Reproduction Laboratory of Ceara State University for the 304 technical support. We would also like to thank Pathovet Laboratory for histopathological 305 analysis and these funding agencies for financial support: FUNCAP (Fundação Cearense de 306 Apoio ao Desenvolvimento Científico e Tecnológico – Ceara’s Foundation of Scientific and 307 Technologic Development Support), CNPq (Conselho Nacional de Desenvolvimento 308 Científico e Tecnológico - National Council for Scientific and Technological Development), 309 CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazilian Federal 310 Agency for the Support and Evaluation of Graduate Education) through research grants 311 (CAPES/Cofecub number 88881.142966/2017-01). 312 313 CONFLICTS OF INTEREST 314 The authors have no competing interests to declare. 315 316 AUTHORS CONTRIBUTIONS 317 DIAT was the counselor; DMSC, MBS, DIAT and BFB designed the methodology; DMSC, 318 MBS, BFB, TGPN, VLT performed the experiment; SST performed handling and 76
319 anaesthesia; DAV performed histopathological processing and analysis; LDMS and DIAT 320 provided equipment and reagents; DMSC drafted the manuscript and DIAT, LDMS, MBS 321 and BFB revised it critically.
322 323 ORCID
324 Dárcio Ítalo Alves Teixeira
325 http://orcid.org/0000-0003-1799-1675
326 Lúcia Daniel Machado da Silva
327 http://orcid.org/0000-0001-9793-1968
328
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501 83
502 TABLE 1. City, age, weight, proved fertility, housing with female and origin of the six gray 503 brocket deer (Mazama gouazoubira). 504 Male City located Age Weight (kg) Proved Housed Origin/born (approximately) fertility with female Male 1 Fortaleza, CE 3 years 14 No No Captive Male 2 Caucaia, CE 5 years 15 Yes Yes Captive Male 3 Caucaia, CE 11 years 15 Yes Yes Captive Male 4 Fortaleza, CE 3 years 15.6 No Yes Captive Male 5 Caucaia, CE 1 year 15 No Yes Captive Male 6 Caucaia, CE 3 years 20 No Yes Captive 505 506 507 TABLE 2. General seminal parameters of normospermic males (F+ group; n = 6 ejaculates) 508 and male 3’s first collection. Values (Mean ± SD). Male 3 (First Seminal parameters F+ collection) Total motility (%) 96.83 ± 3.71 5 Vigor 4.41 ± 0.38 1 Concentration (× 10⁷ 94.08 ± 65.04 4 sptz/ml) 509 510 TABLE 3. Testicular parameters of normospermic (F+) and oligo/azoospermic (F-) gray 511 brocket deer (Mazama gouazoubira). Values (Mean ± SD). Testicular F+ F- parameters Scrotal 14.57 ± 12.06 ± circumference 1.19 a 0.62 b (cm) Testicular volume 26.18 ± 14.99 ± (cm3) 4.94 a 2.66 b 512 Different letters show significant difference between groups (P < 0.05). 513 514 84
515 516 TABLE 4. B mode and spectral Doppler ultrasound parameters of normospermic (F+) and 517 oligo/azoospermic (F-) gray brocket deer (Mazama gouazoubira). Values (Mean ± SD). Ultrasound F+ F- parameters PSV (cm/s) 10.66 ± 5.60 a 9.77 ± 4.71 a EDV (cm/s) 5.59 ± 2.17 a 5.67 ± 2.13 a TAMAX (cm/s) 6.38 ± 2.62 a 7.14 ± 2.77 a PI 0.89 ± 0.75 a 0.53 ± 0.12 a RI 0.40 ± 0.10 a 0.38 ± 0.06 a NPV (longitudinal 66.16 ± 30.66 a 29.42 ± 12.42 a testes) NPV (cross- 69.88 ± 24.00 a 28.26 ± 13.75 b sectional testes) NPV 42.28 ± 14.32 a 24.95 ± 6.78 a (epididymis) PSD (longitudinal 13.92 ± 10.47 a 7.71 ± 3.41 a testes) PSD (cross- 10.78 ± 3.42 a 6.70 ± 1.84 b sectional testes) PSD 12.74 ± 6.59 a 10.77 ± 8.31 a (epididymis) 518 Different letters show significant difference between groups (P < 0.05). 519 520 521 522 523 524 525 526 527 85
528 TABLE 5. Correlations between the ultrasound parameters, scrotal circumference, and 529 testicular volume in gray brocket deer (Mazama gouazoubira). Ultrasound Scrotal circumference Testicular volume parameters PSV (cm/s) r = 0.13, P = 0.71 r = 0.33, P = 0.32 EDV (cm/s) ρ = -0.17, P = 0.61 ρ = 0.16, P = 0.63 TAMAX (cm/s) ρ = -0.20, P = 0.56 ρ = 0.19, P = 0.57 PI ρ = 0.22, P = 0.51 ρ = 0.37, P = 0.26 RI r = 0.44, P = 0.18 r = 0.46, P = 0.15 NPV (longitudinal r = 0.76, P = 0.01* r = 0.78, P = 0.008* testes) NPV (cross-sectional r = 0.89, P = 0.0006* r = 0.91, P = 0.0003* testes) NPV (epididymis) r = 0.50, P = 0.14 r = 0.40, P = 0.26 PSD (longitudinal ρ = 0.44, P = 0.21 ρ = 0.59, P = 0.07 testes) PSD (cross-sectional ρ = 0.80, P = 0.0052 ρ = 0.82, P = 0.004* testes) PSD (epididymis) ρ = 0.14, P = 0.70 ρ = 0.33, P = 0.35 530 * shows significant correlations (P < 0.05). 531 r = Pearson’s correlation coefficient; ρ = Spearman’s correlation coefficient. 532 533 534 535 536 537 538 539 540 541 542 543 544 86
545 Figure legends
546 547 Figure 1. Computer-assisted analysis of the ultrasonographic images of gray brocket deer testes. 548 (A) Position of the transducer for visualizing a longitudinal section of the testis. (B) Position of 549 the transducer for visualizing a cross-sectional section of the testis. (C) Spot-meter technique 550 of pixel analysis: six open squares, 3 × 3 mm2 in size, are placed on a longitudinal section of 551 the ultrasonographic image of the testis. (D) Four open squares, 3 × 3 mm2 in size, are placed 552 on a cross-sectional section of the ultrasonographic image of the testis. (E) Two open squares, 553 3 × 3 mm2 in size, are placed on a longitudinal section of the ultrasonographic image of the 554 epididymis tail. (F) Total pixel number (Count), mean NPV (Mean), standard deviation of NPV 87
555 (StdDev), minimum value of NPV (Min), and maximum value of NPV (Max), most frequently 556 occurring NPV within the selection (Mode) shown on a histogram. 557
558 559 Figure 2. Mode B ultrasonographic images of right (R) or left (L) epididymis (EPID) and testes 560 (TEST) on longitudinal (LONG) or cross-sectional (CS) views. (A-C) Images of F+ males. (D- 561 F) Images of F- males. 562 88
563 564 Figure 3. Waveform of supratesticular artery of gray brocket deer using spectral Doppler 565 technique. 89
8 CONCLUSÕES
A partir dos resultados obtidos nesse estudo, é possível concluir que: a) Os animais normospérmicos apresentaram boa qualidade seminal, com variações individuais nos parâmetros de atividade mitocondrial, morfologia e morfometria espermáticas, demonstrando que diferentes análises seminais podem se complementar e fornecer importantes informações acerca do potencial fértil dos machos de Mazama gouazoubira. b) Maiores circunferência escrotal, volume testicular e intensidade e heterogeneidade de pixels foram detectáveis em animais normospérmicos e correlacionáveis, possivelmente explicado por alterações no parênquima testicular, sem que, porém, influenciassem o fluxo sanguíneo da artéria supratesticular.
90
9 PERSPECTIVA
O presente trabalho fornece informações que auxiliarão em futuras pesquisas sobre conservação de germoplasma utilizando o sêmen ou no desenvolvimento de biotécnicas reprodutivas. Além disso, a utilização de novas técnicas abre novas perspectivas para o diagnóstico de infertilidade e melhor seleção de machos em programas de reprodução assistida para o veado-catingueiro e outras espécies do gênero Mazama sp.
91
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