Impact of adding different concentrations of IGF-I and insulin to the semen extender on bull sperm quality post-cryopreservation / [Impacto da adição de diferentes concentrações de IGF-I e insulina ao diluidor de sêmen sobre a qualidade espermática bovina após a criopreservação]

ABSTRACT This study aimed to evaluate the addition of different concentrations of IGF-I and insulin to egg yolk-based extender to improve bovine semen cryopreservation. Two experiments were developed to evaluate the effects of the additives in two commercial extenders, Botubov® (Experiment 1) and Triladyl® (Experiment 2), both with the same design. Three ejaculates from four bulls (n = 12) were used. Each ejaculate was divided into seven equal fractions for dilution (60x106 spermatozoa/mL) in the following treatments: CON: extender only; IGF100: IGF-I 100ng/mL; IGF200: IGF-I 200ng/mL; INS150: insulin 150µUI/mL; INS200: insulin 200µUI/mL; ASS1: IGF-I 100ng/mL + insulin 150µUI/mL; ASS2: IGF-I 200ng/mL + insulin 200µUI/mL. Semen was cryopreserved by an automated system. Post-thawed sperm were evaluated regarding motility by CASA (Computer-assisted sperm analysis), and membranes by fluorescent probes (H342, PI, FITC-PSA and JC-1). For Botubov® extender, INS150 was more efficient in preserving total and progressive motility, VCL, BCF, plasma and mitochondrial membranes. A similar response was seen when insulin was added to the Triladyl® extender, INS150 was more efficient in preserving sperm motility, plasma membrane integrity and mitochondrial potential. Thus, the addition of insulin 150µUI/mL, regardless of the composition of the extender, contributes to better preserving bovine sperm from the cryopreservation effects.

RESUMO Este estudo teve o objetivo de avaliar a adição de diferentes concentrações de IGF-I e insulina a diluidores, à base de gema de ovo, para melhorar a criopreservação do sêmen bovino. Dois experimentos foram desenvolvidos para avaliar os efeitos dos aditivos em dois diluidores comerciais: Botubov® (Experimento 1) e Triladyl® (Experimento 2), ambos com o mesmo delineamento. Foram utilizados três ejaculados de quatro touros (n=12). Cada ejaculado foi dividido em sete frações para diluição (60x106espermatozoides/mL), nos seguintes tratamentos: CON: somente diluidor; IGF100: 100ng/mL de IGF-I; IGF200: 200ng/mL de IGF-I; INS150: 150µUI/mL de insulina; INS200: 200µUI/mL de insulina; ASS1: 100ng/mL de IGF-I + 150µUI/mL de insulina; ASS2: 200ng/mL de IGF-I + 200µUI/mL de insulina. O sêmen foi criopreservado por sistema automatizado. Após a criopreservação, o sêmen foi avaliado quanto à motilidade espermática por CASA e quanto às membranas espermáticas (plasmática, acrossomal e mitocondrial) por sondas fluorescentes (H342, PI, FITC-PSA e JC-1). Para o diluidor Botubov®, INS150 foi mais eficiente em preservar motilidades total e progressiva, VCL, BCF, integridade da membrana plasmática e potencial mitocondrial. Resposta semelhante foi observada quando a insulina foi adicionada ao diluidor Triladyl®, INS150 foi mais eficiente na preservação da motilidade, integridade das membranas e potencial mitocondrial quando comparado aos demais grupos. Assim, a adição de 150µUI/mL de insulina aos diluidores, independentemente da composição, contribui para melhor criopreservação dos espermatozoides bovinos.

Investigando a fragmentação não induzida e a susceptibilidade à fragmentação do DNA espermático: refinamento da avaliação espermática. Parte 2 / Investigating not induced fragmentation and susceptibility of sperm DNA: refinement of sperm evaluation. Second part

A integridade do DNA espermático está intimamente relacionada com a fertilidade. Técnicas queavaliam o DNA espermático possuem grande importância e potencial de serem aplicadas nas rotinas dasavaliações andrológicas. No entanto, são necessários maiores conhecimentos sobre as técnicas e seus princípiospara permitir que sejam empregadas apropriadamente de acordo com os objetivos e resultados esperados. Dessaforma, assim como abordado na Parte 1, nesta Parte 2 serão abordadas técnicas de avaliação do DNAespermático: TUNEL, laranja de acridina e Sperm Chromatin Structure Assay (SCSA). A técnica de TUNELfundamenta-se na adição de nucleotídeos modificados marcados com fluorescência às fitas fragmentadas. Atécnica avalia, portanto, diretamente a fragmentação de DNA da amostra. A sonda laranja de acridina é capaz dediferenciar, por meio de diferentes colorações, o DNA fragmentado do não fragmentado. Já a técnica de SCSAbaseia-se na imposição de um desafio ao espermatozoide; esta técnica avalia a susceptibilidade dosespermatozoides à fragmentação de DNA, sendo, portanto, uma avaliação indireta. A presente revisão permiteexpor a opinião dos autores sobre as diferentes técnicas e suas aplicações, mostrando o potencial emprego delasnos exames de rotina e nas pesquisas.(AU)

Sperm DNA integrity is directly related to male fertility. Techniques to assess sperm DNA are importantand have potential to be applied in routine evaluation of semen. However, the techniques and principles need tobe further studied to apply they correctly and to get relevant results. Thus, as discussed in Part 1, Part 2 willaddress evaluation techniques of sperm DNA: TUNEL, acridine orange and Sperm Chromatin Structure Assay(SCSA). TUNEL is based on addition of modified nucleotides labeled with fluorescence to fragmented tapesevaluating, therefore, directly, the DNA fragmentation of the sample. Acridine orange differentiates fragmentedDNA by different colors. On the other hand, SCSA are based on the imposition of a challenge to sperm. Thus,this technique assesses the susceptibility of sperm DNA fragmentation constituting indirect assessment. Thereview allows showing opinion of the authors about different techniques and their applications. Therefore,potential use of these techniques in routine exams and researches are addressed.(AU)

Investigando a fragmentação não induzida e a susceptibilidade à fragmentação do DNA espermático: refinamento da avaliação espermática. Parte 2 / Investigating not induced fragmentation and susceptibility of sperm DNA: refinement of sperm evaluation. Second part

A integridade do DNA espermático está intimamente relacionada com a fertilidade. Técnicas queavaliam o DNA espermático possuem grande importância e potencial de serem aplicadas nas rotinas dasavaliações andrológicas. No entanto, são necessários maiores conhecimentos sobre as técnicas e seus princípiospara permitir que sejam empregadas apropriadamente de acordo com os objetivos e resultados esperados. Dessaforma, assim como abordado na Parte 1, nesta Parte 2 serão abordadas técnicas de avaliação do DNAespermático: TUNEL, laranja de acridina e Sperm Chromatin Structure Assay (SCSA). A técnica de TUNELfundamenta-se na adição de nucleotídeos modificados marcados com fluorescência às fitas fragmentadas. Atécnica avalia, portanto, diretamente a fragmentação de DNA da amostra. A sonda laranja de acridina é capaz dediferenciar, por meio de diferentes colorações, o DNA fragmentado do não fragmentado. Já a técnica de SCSAbaseia-se na imposição de um desafio ao espermatozoide; esta técnica avalia a susceptibilidade dosespermatozoides à fragmentação de DNA, sendo, portanto, uma avaliação indireta. A presente revisão permiteexpor a opinião dos autores sobre as diferentes técnicas e suas aplicações, mostrando o potencial emprego delasnos exames de rotina e nas pesquisas.

Sperm DNA integrity is directly related to male fertility. Techniques to assess sperm DNA are importantand have potential to be applied in routine evaluation of semen. However, the techniques and principles need tobe further studied to apply they correctly and to get relevant results. Thus, as discussed in Part 1, Part 2 willaddress evaluation techniques of sperm DNA: TUNEL, acridine orange and Sperm Chromatin Structure Assay(SCSA). TUNEL is based on addition of modified nucleotides labeled with fluorescence to fragmented tapesevaluating, therefore, directly, the DNA fragmentation of the sample. Acridine orange differentiates fragmentedDNA by different colors. On the other hand, SCSA are based on the imposition of a challenge to sperm. Thus,this technique assesses the susceptibility of sperm DNA fragmentation constituting indirect assessment. Thereview allows showing opinion of the authors about different techniques and their applications. Therefore,potential use of these techniques in routine exams and researches are addressed.

Investigando a compactação e a fragmentação não induzida do DNA espermático: refinamento da avaliação espermática parte 1 / Investigating condensation and not induced fragmentation of sperm DNA: Refinement of sperm evaluation first part

O DNA espermático possui papel essencial na reprodução, visto que danos no material genético podemprejudicar os processos de fecundação e desenvolvimento embrionário. Ademais, há evidências de que danos aoDNA podem ser causados por alterações epigenéticas. Entretanto, embora ainda não seja aplicada nas avaliaçõesandrológicas de rotina, faz-se essencial a avaliação do status de integridade do DNA espermático. Existemdiversas técnicas de avaliação com diferentes princípios e objetivos. A aplicação destas irá depender dosequipamentos disponíveis, do treinamento da equipe e das respostas a serem obtidas. Ainda, para a interpretaçãodos resultados obtidos, é necessário o conhecimento do princípio de análise de cada técnica. Assim, a presenterevisão, dividida em duas partes, tem por objetivo abordar diferentes técnicas de avaliação. As técnicasabordadas na parte 1 estão relacionadas às avaliações de compactação da cromatina (azul de toluidina, azul deanilina e cromomicina A3) e à avaliação direta da fragmentação de DNA (ensaio Cometa e teste de dispersão dacromatina modificado). Na parte 2, serão abordadas as técnicas de TUNEL, laranja de acridina e “SpermChromatin Structure Assay” (SCSA). Nesta revisão, serão abordados os princípios dos testes e a interpretaçãodos resultados baseados na literatura e nas pesquisas do grupo.(AU)

Sperm DNA has an important role on reproduction. Injuries in genetic material can impair thefertilization processes and embryonic development. Furthermore, there is evidence that DNA damage can becaused by epigenetic alterations. Although DNA evaluation in sperm analysis routine assessments is essential, itis not yet performed. There are numerous techniques of DNA evaluation with different principles and objectives.Application of these techniques will depend on the equipment available, the staff training and answers to berequired. To interpret the results, however, knowledge of each analysis technique is required. Therefore, thisreview, divided in two parts, aims to consider different techniques. The techniques that will be consideredthroughout the first part of review are: toluidine blue, aniline blue, A3 chromomycyn, Comet assay anddispersion test of modified chromatin. In the second part, it will be approached TUNEL, acridine orange andSperm Chromatin Structure Assay (SCSA) techniques. We will discuss the testes and the main results based onliterature and researches of our group.(AU)

Ferramentas para avaliação da funcionalidade da mitocôndria espermática / Tools for evaluating the functionality of sperm mitochondria

A mitocôndria é a principal fonte de energia para a motilidade e a homeostase espermática. Durante afosforilação oxidativa são produzidos metabólitos, denominados espécies reativas de oxigênio (EROs), as quaisdesempenham papel fundamental nos processos fisiológicos. No entanto, disfunções mitocondriais podem causardesequilíbrio entre a produção de EROs e os mecanismos antioxidantes, provocando o estresse oxidativo, letalpara a célula espermática. Assim, como essa organela está envolvida tanto nos processos fisiológicos quanto nospatológicos dos espermatozoides, fica clara a importância de se avaliar sua funcionalidade. Portanto, o objetivodesta revisão é descrever as possíveis técnicas de avaliação da funcionalidade das mitocôndrias espermáticas pormeio da atividade e do potencial da membrana mitocondrial, da mensuração dos níveis de ATP e ADP e damensuração dos níveis de cálcio.(AU)

Mitochondria are the major source of energy for sperm motility and to the homeostasis. During theoxidative phosphorylation metabolites called Reactive Oxygen Species (ROS), are produced and play a key rolefor the physiological processes. However, mitochondrial dysfunctions can cause an imbalance between ROSproduction and antioxidant mechanisms cause oxidative stress, lethal for spermatozoa. Thus, as this organelle isinvolved both in physiological or pathological processes of sperm, it is clear the importance of evaluating itsfunctionality. Therefore, the aim of this review is to describe the possible techniques for assessing functionalityof sperm mitochondria and these assessments of activity and mitochondrial membrane potential, measuring thelevels of ATP and ADP and measurement the levels of calcium.(AU)

Investigando a compactação e a fragmentação não induzida do DNA espermático: refinamento da avaliação espermática – parte 1 / Investigating condensation and not induced fragmentation of sperm DNA: Refinement of sperm evaluation – first part

O DNA espermático possui papel essencial na reprodução, visto que danos no material genético podemprejudicar os processos de fecundação e desenvolvimento embrionário. Ademais, há evidências de que danos aoDNA podem ser causados por alterações epigenéticas. Entretanto, embora ainda não seja aplicada nas avaliaçõesandrológicas de rotina, faz-se essencial a avaliação do status de integridade do DNA espermático. Existemdiversas técnicas de avaliação com diferentes princípios e objetivos. A aplicação destas irá depender dosequipamentos disponíveis, do treinamento da equipe e das respostas a serem obtidas. Ainda, para a interpretaçãodos resultados obtidos, é necessário o conhecimento do princípio de análise de cada técnica. Assim, a presenterevisão, dividida em duas partes, tem por objetivo abordar diferentes técnicas de avaliação. As técnicasabordadas na parte 1 estão relacionadas às avaliações de compactação da cromatina (azul de toluidina, azul deanilina e cromomicina A3) e à avaliação direta da fragmentação de DNA (ensaio Cometa e teste de dispersão dacromatina modificado). Na parte 2, serão abordadas as técnicas de TUNEL, laranja de acridina e “SpermChromatin Structure Assay” (SCSA). Nesta revisão, serão abordados os princípios dos testes e a interpretaçãodos resultados baseados na literatura e nas pesquisas do grupo.

Sperm DNA has an important role on reproduction. Injuries in genetic material can impair thefertilization processes and embryonic development. Furthermore, there is evidence that DNA damage can becaused by epigenetic alterations. Although DNA evaluation in sperm analysis routine assessments is essential, itis not yet performed. There are numerous techniques of DNA evaluation with different principles and objectives.Application of these techniques will depend on the equipment available, the staff training and answers to berequired. To interpret the results, however, knowledge of each analysis technique is required. Therefore, thisreview, divided in two parts, aims to consider different techniques. The techniques that will be consideredthroughout the first part of review are: toluidine blue, aniline blue, A3 chromomycyn, Comet assay anddispersion test of modified chromatin. In the second part, it will be approached TUNEL, acridine orange andSperm Chromatin Structure Assay (SCSA) techniques. We will discuss the testes and the main results based onliterature and researches of our group.

Ferramentas para avaliação da funcionalidade da mitocôndria espermática / Tools for evaluating the functionality of sperm mitochondria

A mitocôndria é a principal fonte de energia para a motilidade e a homeostase espermática. Durante afosforilação oxidativa são produzidos metabólitos, denominados espécies reativas de oxigênio (EROs), as quaisdesempenham papel fundamental nos processos fisiológicos. No entanto, disfunções mitocondriais podem causardesequilíbrio entre a produção de EROs e os mecanismos antioxidantes, provocando o estresse oxidativo, letalpara a célula espermática. Assim, como essa organela está envolvida tanto nos processos fisiológicos quanto nospatológicos dos espermatozoides, fica clara a importância de se avaliar sua funcionalidade. Portanto, o objetivodesta revisão é descrever as possíveis técnicas de avaliação da funcionalidade das mitocôndrias espermáticas pormeio da atividade e do potencial da membrana mitocondrial, da mensuração dos níveis de ATP e ADP e damensuração dos níveis de cálcio.

Mitochondria are the major source of energy for sperm motility and to the homeostasis. During theoxidative phosphorylation metabolites called Reactive Oxygen Species (ROS), are produced and play a key rolefor the physiological processes. However, mitochondrial dysfunctions can cause an imbalance between ROSproduction and antioxidant mechanisms cause oxidative stress, lethal for spermatozoa. Thus, as this organelle isinvolved both in physiological or pathological processes of sperm, it is clear the importance of evaluating itsfunctionality. Therefore, the aim of this review is to describe the possible techniques for assessing functionalityof sperm mitochondria and these assessments of activity and mitochondrial membrane potential, measuring thelevels of ATP and ADP and measurement the levels of calcium.

Organic selenium supplementation increases PHGPx but does not improve viability in chilled boar semen.

This study evaluated the effects of dietary organic selenium (Se) on viability of chilled boar semen. Twelve boars were divided into three groups: control (CON), 0.3 mg kg(-1) sodium selenite; inorganic (INO), 0.5 mg kg(-1) sodium selenite and organic (ORG), 0.5 mg kg(-1) Se yeast. The experiment was conducted within 10 weeks, and analysis was performed fortnightly, in storage semen by 72 h. No effect was observed on motility; however, straightness and linearity percentages were higher (P < 0.05) in the animals receiving CON diet compared with INO group. Percentages of cells with both plasma and acrosomal intact membranes, lipidic membrane peroxidation and mitochondrial membrane potential were similar on all treatments. Animals receiving CON diet presented higher (P < 0.05) values of ATP when compared with INO group. The PHGPx was higher (P < 0.05) in animals that received ORG in comparison with INO group. In conclusion, organic selenium supplementation increases PHGPx but does not improve chilled semen viability in 72 h.

Use of cholesterol-loaded cyclodextrin in donkey semen cryopreservation improves sperm viability but results in low fertility in mares.

The use of cholesterol-loaded cyclodextrin (CLC) on semen cryopreservation has been related with better sperm viability in several species; however, the effect on fertility is not known in donkey semen. Ejaculates (n = 25) from five donkeys were diluted in S-MEDIUM with 0, 1, 2 or 3 mg of CLC/120 × 10(6) spermatozoa. Semen was frozen, and thawed samples were evaluated by computer-assisted sperm analyser system (CASA), supravital test, hyposmotic swelling test and fluorescent dyes to assess the integrity of sperm membranes. Mares (n = 60) were inseminated with frozen-thawed semen treated with the doses of 0 or 1 mg CLC. Percentages of sperm with progressive motility and with functional plasma membrane were greater (p < 0.05) in the CLC-treated groups than in the control. Percentages of intact plasma membrane and intact plasma membrane and acrosome detected by fluorescent dyes were also greater (p < 0.05) in CLC-treated groups. Although no difference (p > 0.05) in conception rates was detected between groups (control, 3/30, 10%; CLC-treated, 1/30, 3.3%), fertility was low for artificial insemination programs in mares. Therefore, we firstly demonstrated that frozen semen treated with CLC in S-MEDIA extender before freezing improves the in vitro sperm viability, but semen treated or not with CLC in S-MEDIUM extender results in a very low conception rate in mares inseminated with thawed donkey semen.

Heparin and penicillamine-hypotaurine-epinephrine (PHE) solution during bovine in vitro fertilization procedures impair the quality of spermatozoa but improve normal oocyte fecundation and early embryonic development.

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P<0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P<0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P>0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P>0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P>0.05) in the presence of heparin and PHE (P<0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development.

O uso de ECG influencia a taxa de concepção em vacas Nelore de diferentes condições corporais submetidas ao mesmo protocolo de IATF? / Does ECG influence the conception rate Nelore cows presenting different body condition scores submitted to the same timed-AI protocol?

O objetivo deste estudo foi avaliar a taxa de concepção (TC) de vacas multíparas da raça Nelore apresentando diferentes escores de condição corporal (ECC), que foram submetidas ao mesmo protocolo de inseminação artificial em tempo fixo (IATF) com o emprego da gonadotrofina coriônica equina (eCG). Foram inseminadas 1574 vacas da raça Nelore, com 40 a 50 dias pós-parto. Durante as IATFs, os dados referentes ao touro (n = 8), inseminador (n = 3) e ECC foram anotados, sendo a nota do ECC aferida de 1 a 5 por técnico experiente. O diagnóstico de gestação foi realizado, por ultrassonografia, 40 dias após a IATF. Não foi observado efeito (P > 0,05) de inseminador ou touro na TC. Também não se observou diferença estatística desta taxa entre os grupos de animais separados de acordo com os ECCs. O grupo de animais com menor ECC (Grupo 1 = ECC 1,5 a 2,0; n = 139) apresentou TC de 47,4%. Os grupos de animais com ECC entre 2,5 a 2,75 (Grupo 2; n = 741) e com ECC entre 3,0 a 3,25 (Grupo 3; n = 463) apresentaram, respectivamente, TC de 47,6% e 51,2%. Os grupos de animais com maior ECC (Grupo 4 = ECC 3,5 a 4,0; n = 231) apresentou TC de 45,3% (P > 0,05). Concluiu-se que as taxas de concepção foram semelhantes entre os animais apresentando diferentes ECC no rebanho, provavelmente pelo eCG compensar a baixa pulsatilidade de LH dos animais mais magros. Porém, recomenda-se maiores estudos para verificar a real necessidade do uso de eCG em animais de condição corporal superior a 3,5.

The aim of this study was to evaluate the conception rate (CR) of multiparous Nelore cows presenting different body condition scores (BCS), which were submitted to the same Timed-AI protocol with equine chorionic gonadotrophin (eCG). A total of 1574 cows were inseminated, between 40 and 50 days postpartum. During insemination (timed-AI), all data regarding to bull (n=8), inseminator (n=3) and BCS (1 to 5) were recorded. The pregnancy diagnosis was performed, by ultrasonography, 40 days after timed-AI. No effect (P>0.05) of inseminator or bull was observed. No statistical difference was also observed between the groups of animals with different BCS. The animals with lower BCS (Group 1 = BCS 1.5 to 2.0; n = 139) had a CR of 47.4%. The animals with BCS from 2.5 to 2.75 (Group 2; n = 741) and BCS from 3.0 to 3.25 (Group 3; n = 463) had a CR of 47.6% and 51.2%, respectively. The animals with higher BCS (Group 4 = BCS 3.5 to 4.0; n = 231) had a CR of 45.3% (P > 0.05). It was concluded that conception rates were similar between the animals presenting different BCS in the herd, likely because the eCG minimized the effects of low LH pulsatility in animals presenting reduced nutritional condition. However, other studies are recommended to verify the real need of using eCG in animals with body condition exceeding 3.5.

O uso de ECG influencia a taxa de concepção em vacas Nelore de diferentes condições corporais submetidas ao mesmo protocolo de IATF? / Does ECG influence the conception rate Nelore cows presenting different body condition scores submitted to the same timed-AI protocol?

O objetivo deste estudo foi avaliar a taxa de concepção (TC) de vacas multíparas da raça Nelore apresentando diferentes escores de condição corporal (ECC), que foram submetidas ao mesmo protocolo de inseminação artificial em tempo fixo (IATF) com o emprego da gonadotrofina coriônica equina (eCG). Foram inseminadas 1574 vacas da raça Nelore, com 40 a 50 dias pós-parto. Durante as IATFs, os dados referentes ao touro (n = 8), inseminador (n = 3) e ECC foram anotados, sendo a nota do ECC aferida de 1 a 5 por técnico experiente. O diagnóstico de gestação foi realizado, por ultrassonografia, 40 dias após a IATF. Não foi observado efeito (P > 0,05) de inseminador ou touro na TC. Também não se observou diferença estatística desta taxa entre os grupos de animais separados de acordo com os ECCs. O grupo de animais com menor ECC (Grupo 1 = ECC 1,5 a 2,0; n = 139) apresentou TC de 47,4%. Os grupos de animais com ECC entre 2,5 a 2,75 (Grupo 2; n = 741) e com ECC entre 3,0 a 3,25 (Grupo 3; n = 463) apresentaram, respectivamente, TC de 47,6% e 51,2%. Os grupos de animais com maior ECC (Grupo 4 = ECC 3,5 a 4,0; n = 231) apresentou TC de 45,3% (P > 0,05). Concluiu-se que as taxas de concepção foram semelhantes entre os animais apresentando diferentes ECC no rebanho, provavelmente pelo eCG compensar a baixa pulsatilidade de LH dos animais mais magros. Porém, recomenda-se maiores estudos para verificar a real necessidade do uso de eCG em animais de condição corporal superior a 3,5. (AU)

The aim of this study was to evaluate the conception rate (CR) of multiparous Nelore cows presenting different body condition scores (BCS), which were submitted to the same Timed-AI protocol with equine chorionic gonadotrophin (eCG). A total of 1574 cows were inseminated, between 40 and 50 days postpartum. During insemination (timed-AI), all data regarding to bull (n=8), inseminator (n=3) and BCS (1 to 5) were recorded. The pregnancy diagnosis was performed, by ultrasonography, 40 days after timed-AI. No effect (P>0.05) of inseminator or bull was observed. No statistical difference was also observed between the groups of animals with different BCS. The animals with lower BCS (Group 1 = BCS 1.5 to 2.0; n = 139) had a CR of 47.4%. The animals with BCS from 2.5 to 2.75 (Group 2; n = 741) and BCS from 3.0 to 3.25 (Group 3; n = 463) had a CR of 47.6% and 51.2%, respectively. The animals with higher BCS (Group 4 = BCS 3.5 to 4.0; n = 231) had a CR of 45.3% (P > 0.05). It was concluded that conception rates were similar between the animals presenting different BCS in the herd, likely because the eCG minimized the effects of low LH pulsatility in animals presenting reduced nutritional condition. However, other studies are recommended to verify the real need of using eCG in animals with body condition exceeding 3.5. (AU)

Effect of sequence of insemination after simultaneous thawing of multiple semen straws on conception rate to timed AI in suckled multiparous Nelore cows.

The objective was to determine the effect of sequence of insemination after simultaneous thawing of multiple 0.5 mL semen straws on conception rate in suckled multiparous Nelore cows. The effect of this thawing procedure on in vitro sperm characteristics was also evaluated. All cows (N = 944) received the same timed AI protocol. Ten straws (0.5 mL) of frozen semen from the same batch were simultaneously thawed at 36 °C, for a minimum of 30 sec. One straw per cow was used for timed AI. Frozen semen from three Angus bulls was used. Timed AI records included sequence of insemination (first to tenth) and time of semen removal from thawing bath. For laboratory analyses, the same semen batches used in the field experiment were evaluated. Ten frozen straws from the same batch were thawed simultaneously in a thawing unit identical to that used in the field experiment. The following sperm characteristics were analyzed: sperm motility parameters, sperm thermal resistance, plasma and acrosomal membrane integrity, lipid peroxidation, chromatin structure, and sperm morphometry. Based on logistic regression, there were no significant effects of breeding group, body condition score, AI technician, and sire on conception rate, but there was an interaction between sire and straw group (P = 0.002). Semen from only one bull had decreased (P < 0.05) field fertility for the group of straws associated with the longest interval from thawing to AI. However, the results of the laboratory experiment were unable to explain the findings of the field experiment. Sperm width:length ratio of morphometric analysis was the single sperm characteristic with a significant interaction between sire and straw group (P = 0.02). It was concluded that sequence of insemination after simultaneous thawing of 10 semen straws can differently affect conception rates at timed AI, depending on the sire used. Nevertheless, the effects of this thawing environment on in vitro sperm characteristics, remain to be further investigated.

Effects of bovine sperm cryopreservation using different freezing techniques and cryoprotective agents on plasma, acrosomal and mitochondrial membranes.

The success of semen cryopreservation is influenced by several factors, such as freezing curves and cryoprotectants. These two factors are of special interest once they may lead to many important physical-chemical changes resulting in different degrees of damage in spermatozoa structure. This experiment was designed to compare the effect of bull semen cryopreservation using two freezing techniques: conventional (CT--cooling rate of -0.55 °C min(-1) and freezing rate of -19.1 °C min(-1) and automated (AT--cooling rate of -0.23 °C min(-1) and freezing rate of -15 °C min(-1)), performed with different curves, and with three cryoprotectants (glycerol, ethylene glycol and dimethyl formamide) on bovine sperm motility and integrity of plasma, acrosomal and mitochondrial membranes. These variables were simultaneously evaluated using the fluorescence probes propidium iodide, fluorescein-conjugated Pisum sativum agglutinin and MitoTracker Green FM. The effects of freezing techniques, as well as of different cryoprotectants were analysed by the analysis of variance. The means were compared by Fisher's test. There were no significant differences between freezing techniques (P > 0.05). Glycerol showed higher percentages of motility, vigour and integrity of plasma, acrosomal and mitochondrial membranes than other two cryoprotectants (P < 0.05). Ethylene glycol preserved higher motility and integrity of plasma and mitochondrial membranes than dimethyl formamide (P < 0.05). Sperm motility with glycerol was 30.67 ± 1.41% and 30.50 ± 1.06%, with ethylene glycol was 21.17 ± 1.66% and 21.67 ± 1.13% and with dimethyl formamide was 8.33 ± 0.65% and 9.17 ± 0.72% to CT and AT curves, respectively. The percentage of spermatozoa with simultaneously intact plasma membrane, intact acrosome and mitochondrial function (IPIAH) was 14.82 ± 1.49% (CT) and 15.83 ± 1.26% (AT) to glycerol, 9.20 ± 1.31% (CT) and 9.92 ± 1.29% (AT) to ethylene glycol 4.65 ± 0.93% (CT) and 5.17 ± 0.87% (AT) to dimethyl formamide. Glycerol provided the best results, although nearly 85% of spermatozoa showed some degree of injury in their membranes, suggesting that further studies are required to improve the results of cryopreservation of bovine semen.

Effects of discontinuous Percoll gradient centrifugation on the quality of bovine spermatozoa evaluated with computer-assisted semen analysis and fluorescent probes association.

The objective of this study was to evaluate the quality of bovine frozen-thawed sperm cells after Percoll gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Four ejaculates per bull were evaluated before and after discontinuous Percoll gradient centrifugation. Sperm motility was assessed by computer-assisted semen analysis and the integrity of the plasma and acrosomal membranes, as well as mitochondrial function, were evaluated using a combination of fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. The procedure of Percoll gradient centrifugation increased the percentage of total and progressive sperm motility, beat frequency, rectilinear motility, linearity and rapidly moving cells. In addition, the percentage of cells with intact plasma membrane and mitochondrial membrane potential was increased in post-centrifugation samples. However, the percentage of sperm cells with intact acrosomal membrane was markedly reduced. The method used selected the motile cells with intact plasma membrane and higher mitochondrial functionality in frozen-thawed bull semen, but processing, centrifugation and/or the Percoll medium caused damage to the acrosomal membrane.

Aspects related to the technique and the utilization of sexed semen in vivo and in vitro

The development of methods capable of selecting the sex of animals has always been a great challenge for humankind. Separating X chromosome- bearing and Y chromosome-bearing sperm based on DNA difference using flow cytometry is the only technique that has achieved a useful level of progress, especially in cattle. This allowed the technique to be commercially applicable in this species. Thorough this review, aspects related to the sexing technique (flow cytometry) and to the sperm will be discussed along with the in vitro use of sexed semen. Besides, number of sperm used and semen deposition location, the influence of reproductive status (heifers vs. cows), time of insemination and production of embryos in superovulated donors will be reviewed.

Aspects related to the technique and the utilization of sexed semen in vivo and in vitro

The development of methods capable of selecting the sex of animals has always been a great challenge for humankind. Separating X chromosome- bearing and Y chromosome-bearing sperm based on DNA difference using flow cytometry is the only technique that has achieved a useful level of progress, especially in cattle. This allowed the technique to be commercially applicable in this species. Thorough this review, aspects related to the sexing technique (flow cytometry) and to the sperm will be discussed along with the in vitro use of sexed semen. Besides, number of sperm used and semen deposition location, the influence of reproductive status (heifers vs. cows), time of insemination and production of embryos in superovulated donors will be reviewed.(AU)

Natural, but not lyophilized, low density lypoproteins were an acceptable alternative to egg yolk for cryopreservation of ram semen.

The objective was to evaluate the suitability of using natural or lyophilized low density lipoproteins (LDL), in lieu of whole egg yolk, in extenders for cryopreserving ram semen. Once extragonadal sperm reserves were depleted in 10 fertile Santa Inês cross rams, two ejaculates per ram were collected for cryopreservation. Nine extenders were used: Tris-16% egg yolk extender with 5% glycerol as a control (T1), and substitution of whole egg yolk with 8, 12, 16 or 20% natural LDL (T2-T5, respectively), or with 8, 12, 16, or 20% lyophilized LDL (T6-T9). Semen was diluted to 100 × 10(6) sperm/mL, packaged into 0.25 mL straws, cooled, held at 5 °C for 3 h, and then frozen in liquid nitrogen vapor. Immediately after thawing (37 °C for 30 s), sperm total and progressive motility, and kinetic parameters were analyzed with computer assisted semen analysis (CASA). Percentage of sperm with plasma membrane functional integrity was assessed by the hypoosmotic swelling test (HOST), sperm membrane physical integrity with propidium iodide (PI), and acrosome integrity with FITC-PSA using an epifluorescent microscope. For all sperm end points, there was no difference between the control and natural LDL treatments (P > 0.05): total motility (T1: 20.9 ± 11.9 and average of T2-T5: 25.9 ± 13.6%; mean ± SD), progressive motility (T1: 6.6 ± 4.2 and average of T2-T5: 11.7 ± 7.5%), HOST(+) (T1: 23.7 ± 6.9 and average of T2-T5: 23.2 ± 8.7 %) and PI(-)/PSA(-) (T1: 13.8 ± 7.8 and average of T2-T5: 18.1 ± 7.8%). However, lyophilization was apparently unable to preserve the protective function of LDL; every sperm end point was significantly worse than in the control and natural LDL groups. We concluded that natural LDL was appropriate for cryopreserving ram semen, as it yielded results similar to those obtained with whole egg yolk.

Addition of seminal plasma to post-thawing equine semen: what is the effect on sperm cell viability?

Effect of seminal plasma addition after thawing on viability or cryocapacitation is not definitively established. This experiment was performed to verify the effect of adding seminal plasma, autologous or homologous (from an animal with good semen freezability). Five ejaculates from each of four stallions with proven fertility were collected and cryopreserved. The semen was subsequently thawed and divided into the following three treatment groups: no seminal plasma addition after semen thawing (NOSP); the addition of homologous seminal plasma after semen thawing (HSP) and the addition of autologous seminal plasma after semen thawing (ASP). The addition of 20% of seminal plasma led to an increase in the cell population that simultaneously show plasma and acrosomal membrane integrity (p < 0.05). The addition of seminal plasma did not alter the total motility, the amount of cells with mitochondrial membrane potential or the sperm velocities (average path velocity, straight-line velocity and curvilinear velocity). However, the beat/cross-frequency, straightness and linearity were reduced in ASP and HSP groups (p < 0.05). Unexpectedly, the addition of homologous seminal plasma reduced the proportion of cells with progressive motility (p < 0.05) and the addition of autologous seminal plasma reduced the amplitude of the lateral head displacement (p < 0.05). Based on the increase in the cell populations that had the plasma and acrosomal membrane integrity simultaneously identified in this study, we proposed that the addition of seminal plasma (autologous or homologous) into post-thawed semen before insemination could increase semen fertility.

Métodos de avaliação da morfologia e função espermática: momento atual e desafios futuros / Methods of the assessment of morphology and function of sperm: actual moment and future challenges

Com o propósito de obter resultados que demonstrem maior repetibilidade na avaliação da morfologia tanto quanto na função espermática, diversas técnicas laboratoriais tem sido desenvolvidas. Surgiram sistemas que utilizam a análise computadorizada de imagens, métodos de “coloração” empregando corantes fluorescentes (sondas fluorescentes) em microscopia de epifluorescência ou citometria de fluxo, que aumentaram a possibilidade de uma análise mais criteriosa da integridade estrutural dos espermatozóides. Os autores descrevem experiências, por mais de uma década, com a utilização destas biotécnicas, fazendo uma análise crítica, bem como apontando os desafios futuros, inclusive na área de biologia molecular espermática.(AU)

In order to obtain results showing greater repeatability in morphology and sperm function assessment, several laboratorial techniques have been developed continuously. Systems that utilize computerized image analysis, staining methods using fluorescent dies (fluorescent probes) in epifluorescence microscopy or flow cytometry, that allowed a more judicious evaluation of sperm structure integrity arose. Authors describe experiences, over a decade, with the use of these biotechiques, critically analyzing and pointing out future challenges, including the field of sperm molecular biology.(AU)