Relationship between sperm ubiquitination and equine semen freezability.

This study aimed to assess the semen ubiquitin levels of stallions with good (GF) and poor semen freezability (PF) and to evaluate the relationship between sperm ubiquitination and sperm morphological defects. Five ejaculates from eight adult stallions (n = 40) were collected and cryopreserved. Then, the ubiquitin level in equine sperm cells was assessed by immunohistochemistry with epifluorescence microscopy, and sperm morphology was assessed by differential interference contrast microscopy. Sperm cells were classified according to the intensity (classification 1: from I to IV; I = very low ubiquitin intensity and IV = very high ubiquitin intensity) and location of ubiquitin staining (classification 2). Statistical analyses were performed using SAS software (version 9.4), and p ≤ .05 was considered significant. We observed that PF stallions showed higher percentages (p < .05) of sperm cells with high ubiquitination (11.82% of ubiquitin intensity grade I, 39.13% of ubiquitin intensity grade II, 27.25% of ubiquitin intensity grade III, and 20.67% of grade IV), while GF stallions showed higher percentages (p < .05) of sperm cells with lower staining intensity (28.52% grade I, 59.83% grade II, 7.92% grade III, and 7.02% grade IV). Furthermore, for PF stallions, 23 significant correlations were detected (p < .05) between sperm abnormalities and ubiquitin intensity in different sperm regions. Increased ubiquitination of the sperm head, midpiece, and tail was positively correlated with their respective morphological defects. We concluded that high sperm ubiquitin levels are observed in ejaculates from stallions with poor semen quality (poor freezability), and ubiquitin marking in specific cellular locations can identify sperm morphological defects.

Using transrectal ultrasound-guided massage of the accessory sex glands for buck semen collection yields semen with greater cryoresistance than electroejaculation alone during the breeding season.

The cryoresistance of bucks' semen collected by transrectal ultrasound-guided massage of the accessory sex glands (TUMASG) and electroejaculation (EE) was compared during the breeding season (Study 1) or the late non-breeding season (Study 2). Semen was collected from 10 Gabon bucks with both methods in each season and then frozen according to a standardized protocol. The time required for ejaculation, the number of electrical pulses applied, the sum of the electrical pulses applied∗voltage and the total number of vocalizations emitted during the collection by each animal were recorded. In Study 1, TUMASG required a longer time (P = 0.0006) but fewer electrical pulses and a lower sum of pulses∗voltage than EE (P < 0.0001 for both comparisons). Bucks vocalized fewer times during TUMASG than during EE (P < 0001). Semen collected with TUMASG had greater sperm concentration, sperm mass motility, total number of motile sperm and of sperm with progressive motility, and tended to have greater total number of sperm with functional membrane than semen collected with EE (P = 0.02; P = 0.003; P = 0.02; P = 0.02; P = 0.06, respectively). After the freezing-thawing process, sperm collected with TUMASG had a greater quality of the motility, a total number of motile sperm and sperm with progressive motility, a greater percentage of motile sperm and sperm with functional membrane, and tended to have a greater total number of sperm with normal morphology than semen collected with EE (P = 0.04; P = 0.04; P = 0.03; P = 0.02; P = 0.04; P = 0.06, respectively). In Study 1 sperm collected with TUMASG had greater cryoresistance for almost all the variables considered than when it was collected with EE. In Study 2, the number of electrical pulses and the sum of pulses∗voltage were greater with EE than with TUMASG (P < 0001; P = 0.0002, respectively). There were no differences in any sperm variable in fresh or thawed samples collected with TUMASG or EE. However, the other seminal characteristics evaluated did not differ according to the method, and there were no differences in the cryoresistance of any variable. Overall, TUMASG affected animal welfare less than EE and was suitable for collecting semen of good quality with high cryoresistance during the breeding season in buck.

Semen quality and freezability analysis during breeding and non-breeding seasons in heavy draft stallions in southern Chile.

The aim of this study was to evaluate seasonal changes in basic parameters of sperm quality and freezability behaviour of ejaculates from 10 fertile heavy draft stallions. A total of 140 ejaculates were collected, processed and evaluated during both the breeding (September-November) and non-breeding seasons (April-June). Fresh semen was evaluated for volume, concentration, total spermatozoa per ejaculate, plasma membrane integrity and total sperm motility. Cryopreserved samples were evaluated for plasma membrane integrity and sperm motility by the CASA system, and for the freezability index (FI), which was defined as the decreased ratio of viability after freezing-thawing. In fresh ejaculates, only viability showed significantly higher values in the breeding than in the non-breeding season (64.0% ± 15.0% vs. 58.6% ± 12.0%, respectively; p < .05). The sperm post-thawing analysis of viability and total motility parameters showed no significant changes linked to the season. However, the FI analysis showed that the ejaculates collected in the non-breeding season had higher cryoresistance characteristics than those collected in the breeding season. Results suggest that the presence of some cryoprotective factor/s in heavy draft horse ejaculates could be modulated by seasonality, with higher protective effects in the non-breeding season.

Differential abundances of four forms of Binder of SPerm 1 in the seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability.

The Binder of SPerm 1 (BSP1) protein is involved in the fertilization and semen cryopreservation processes and is described to be both beneficial and detrimental to sperm. Previously, the relationship of BSP1 with freezability events has not been completely understood. The objective of this work was to determine the differential abundance of the forms of the BSP1 protein in cryopreserved seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability using proteomics. A wide cohort of adult bulls with high genetic value from an artificial insemination center was used as donors of high quality, fresh semen. Nine bulls presenting different patterns of semen freezability were selected. Two-dimensional gel electrophoresis showed differential abundance in a group of seven protein spots in the frozen/thawed seminal plasma from the bulls, ranging from 15 to 17 kDa, with pI values from 4.6 to 5.8. Four of these spots were confirmed to be BSP1 using mass spectrometry, proteomics, biochemical, and computational analysis (Tukey's test at P < 0.05). The protein spot weighing 15.52 ± 0.53 kDa with a pI value of 5.78 ± 0.12 is highlighted by its high abundance in bulls with low semen freezability and its absence in bulls presenting high semen freezability. This is the first report showing that more than two forms of BSP1 are found in the seminal plasma of Nelore adult bulls and not all animals have a similar abundance of each BSP1 form. Different BSP1 forms may be involved in different events of fertilization and the cryopreservation process.

Cisteína como antioxidante para a criopreservação de sêmen de garanhões considerado de baixa congelabilidade / Cysteine as an antioxidant to frozen semen of “bad freezer” stallions

Objetivou-se avaliar o efeito da adição de cisteína (CIS), como agente antioxidante, no diluenteutilizado para criopreservação de espermatozoides equinos com histórico de sêmen com baixa congelabilidade.Três amostras seminais de sete garanhões foram obtidas por meio de vagina artificial, diluídas em meiocomercial (Botusêmen) e distribuídas em dois tratamentos: controle e CIS (2,5 mM de cisteína) ecriopreservadas. Após o descongelamento, as avaliações in vitro foram: cinética espermática computadorizada eavaliação da integridade das membranas plasmática e acrossomal por meio de sondas fluorescentes. Verificou-seque no grupo CIS o percentual de motilidade total e progressiva (23,95 ± 3,33; 4,66 ± 0,84), respectivamente, foisuperior em relação às amostras do grupo controle (11,38 ± 2,35; 2,04 ± 0,36), (P < 0,01). Não houve diferençacom relação à integridade das membranas plasmática (12,5 ± 2,6 x 15,1 ± 2,6) e acrossomal (24,2 ± 3,6 x 25,4 ±3,85), (P > 0,05), quando se comparou grupo controle e cisteína, respectivamente. Levando-se em consideraçãoque a motilidade apresenta alta correlação com a fertilidade, os resultados sugerem que a adição de cisteína naconcentração de 2,5 mM ao meio diluente antes do congelamento de sêmen incrementa a qualidade dosespermatozoides criopreservados de animais considerados de baixa congelabilidade.(AU)

The aim of this experiment was to evaluate the effect of in vitro addition of cysteine (CIS) as antioxidantused for frozen semen of "bad freezer" stallions. Three semen samples from seven stallions were obtained byartificial vagina, diluted in commercial extender (Botusêmen) and splited in two treatments: control and CIS(2.5 mM of cysteine) and cryopreserved. After post-thaw, the analyzed parameters were: computerized analysisof sperm movement characteristics and acrosmal and plasma membrane integrity by fluorescent probes. Totaland progressive motility were higher (P < 0.01) in CIS group (23.95 ± 3.33; 4.66 ± 0.84) when compared withcontrol group (11.38 ± 2.35; 2.04 ± 0.36), respectively. Regarding plasma and acrosomal membrane integritythere was no difference (P > 0.05) (12.5 ± 2.6 x 15.1 ± 2.6 and 24.2 ± 3.6 x 25.4 ± 3.85, respectively) betweenthe control and cysteine group. It is known that the motility is highly correlated with fertility so these resultssuggest that the addition of 2.5 mM of cysteine in the frozen extender medium before cryopreservation increasesthe seminal quality of "bad freezer" stallions.(AU)

Cisteína como antioxidante para a criopreservação de sêmen de garanhões considerado de baixa congelabilidade / Cysteine as an antioxidant to frozen semen of “bad freezer” stallions

Objetivou-se avaliar o efeito da adição de cisteína (CIS), como agente antioxidante, no diluenteutilizado para criopreservação de espermatozoides equinos com histórico de sêmen com baixa congelabilidade.Três amostras seminais de sete garanhões foram obtidas por meio de vagina artificial, diluídas em meiocomercial (Botusêmen) e distribuídas em dois tratamentos: controle e CIS (2,5 mM de cisteína) ecriopreservadas. Após o descongelamento, as avaliações in vitro foram: cinética espermática computadorizada eavaliação da integridade das membranas plasmática e acrossomal por meio de sondas fluorescentes. Verificou-seque no grupo CIS o percentual de motilidade total e progressiva (23,95 ± 3,33; 4,66 ± 0,84), respectivamente, foisuperior em relação às amostras do grupo controle (11,38 ± 2,35; 2,04 ± 0,36), (P 0,05), quando se comparou grupo controle e cisteína, respectivamente. Levando-se em consideraçãoque a motilidade apresenta alta correlação com a fertilidade, os resultados sugerem que a adição de cisteína naconcentração de 2,5 mM ao meio diluente antes do congelamento de sêmen incrementa a qualidade dosespermatozoides criopreservados de animais considerados de baixa congelabilidade.

The aim of this experiment was to evaluate the effect of in vitro addition of cysteine (CIS) as antioxidantused for frozen semen of "bad freezer" stallions. Three semen samples from seven stallions were obtained byartificial vagina, diluted in commercial extender (Botusêmen) and splited in two treatments: control and CIS(2.5 mM of cysteine) and cryopreserved. After post-thaw, the analyzed parameters were: computerized analysisof sperm movement characteristics and acrosmal and plasma membrane integrity by fluorescent probes. Totaland progressive motility were higher (P 0.05) (12.5 ± 2.6 x 15.1 ± 2.6 and 24.2 ± 3.6 x 25.4 ± 3.85, respectively) betweenthe control and cysteine group. It is known that the motility is highly correlated with fertility so these resultssuggest that the addition of 2.5 mM of cysteine in the frozen extender medium before cryopreservation increasesthe seminal quality of "bad freezer" stallions.

Efeitos do diluidor na viabilidade espermática pós-criopreservação em tourinhos da raça Gir Leiteiro aos 25 meses de idade, pré-selecionados pela classificação andrológica por pontos / Effects of the extender in the semen post - thaw viability in young dairy Gyr bulls (Bos taurus indicus), pre - selected by breeding soundness evaluation

Procedeu-se à criopreservação do sêmen de oito tourinhos Gir Leiteiro, com idade média de 25 meses, pré-selecionados para elevada pontuação (média 84,4±5,6) na classificação andrológica por pontos (CAP), em dois diferentes diluidores: um à base de lactose-gema-glicerol e outro à base de lecitina de soja. As curvas de resfriamento e congelação foram padronizadas com o auxílio da máquina CRYOGEN®. Os parâmetros pós-congelação avaliados no sêmen submetido aos dois diluidores - motilidade, vigor, defeitos maiores, menores e totais, defeitos de acrossoma, cauda dobrada, reação ao teste hiposmótico (Thos) e células normais - foram comparados aos do sêmen fresco, exceto para Thos e entre eles. O sêmen de todos os animais foi congelado com êxito no diluidor lactose-gema-glicerol. Houve diferença (P<0,05) em todas as variáveis analisadas no sêmen fresco e pós-congelado, exceto para defeitos maiores. Entre diluidores, houve diferença (P<0,05) para motilidade, vigor, cauda dobrada e Thos. Estes resultados indicam que a seleção pelo CAP médio >80 é um bom índice para selecionar touros com maior taxa de espermatozoides viáveis pós-congelação.(AU)

Semen cryopreservation from eight young dairy Gyr bulls was performed using two different semen extenders. Bulls aging 25 months old and pre-selected for a high average score (84.4±5.6 in a 0-100 scale) in Zebu breeding soundness evaluation (BSE) composed the experimental group. Extenders were based on lactosis-egg yolk-glycerol and soya lecithin. Chilling and freezing curves were standardized by CRYOGEN® machine. Post-thaw features evaluated in semen frost in both extenders - motility, vigor, major, minor and total deffects, morphological alteration in acrossome, bent tail, reative cells to hyposmotic sweeling test (Thos), and normal cells - were compared to the ones in the fresh ejaculate (except Thos) and among them. It was possible to freeze semen from all animals in the lactosis-egg yolk-glycerol extender. There were difference (P<0.05) in all analyzed features between fresh and cryopreserved semen, except for major deffects. Between extenders, there were differences (P<0.05) in motility, vigor, bent tail, and Thos. All bulls had successfull semen freezability. These results sustain that pre-selection for high BSE values (average >80 points) is a good index to identify bulls with good post semen - thaw features. However, the choice of the extender is critical for obtaining acceptable results.(AU)

Efeitos do diluidor na viabilidade espermática pós-criopreservação em tourinhos da raça Gir Leiteiro aos 25 meses de idade, pré-selecionados pela classificação andrológica por pontos / Effects of the extender in the semen post - thaw viability in young dairy Gyr bulls (Bos taurus indicus), pre - selected by breeding soundness evaluation

Procedeu-se à criopreservação do sêmen de oito tourinhos Gir Leiteiro, com idade média de 25 meses, pré-selecionados para elevada pontuação (média 84,4±5,6) na classificação andrológica por pontos (CAP), em dois diferentes diluidores: um à base de lactose-gema-glicerol e outro à base de lecitina de soja. As curvas de resfriamento e congelação foram padronizadas com o auxílio da máquina CRYOGEN®. Os parâmetros pós-congelação avaliados no sêmen submetido aos dois diluidores - motilidade, vigor, defeitos maiores, menores e totais, defeitos de acrossoma, cauda dobrada, reação ao teste hiposmótico (Thos) e células normais - foram comparados aos do sêmen fresco, exceto para Thos e entre eles. O sêmen de todos os animais foi congelado com êxito no diluidor lactose-gema-glicerol. Houve diferença (P<0,05) em todas as variáveis analisadas no sêmen fresco e pós-congelado, exceto para defeitos maiores. Entre diluidores, houve diferença (P<0,05) para motilidade, vigor, cauda dobrada e Thos. Estes resultados indicam que a seleção pelo CAP médio >80 é um bom índice para selecionar touros com maior taxa de espermatozoides viáveis pós-congelação.

Semen cryopreservation from eight young dairy Gyr bulls was performed using two different semen extenders. Bulls aging 25 months old and pre-selected for a high average score (84.4±5.6 in a 0-100 scale) in Zebu breeding soundness evaluation (BSE) composed the experimental group. Extenders were based on lactosis-egg yolk-glycerol and soya lecithin. Chilling and freezing curves were standardized by CRYOGEN® machine. Post-thaw features evaluated in semen frost in both extenders - motility, vigor, major, minor and total deffects, morphological alteration in acrossome, bent tail, reative cells to hyposmotic sweeling test (Thos), and normal cells - were compared to the ones in the fresh ejaculate (except Thos) and among them. It was possible to freeze semen from all animals in the lactosis-egg yolk-glycerol extender. There were difference (P<0.05) in all analyzed features between fresh and cryopreserved semen, except for major deffects. Between extenders, there were differences (P<0.05) in motility, vigor, bent tail, and Thos. All bulls had successfull semen freezability. These results sustain that pre-selection for high BSE values (average >80 points) is a good index to identify bulls with good post semen - thaw features. However, the choice of the extender is critical for obtaining acceptable results.

Proteínas do plasma seminal relacionadas a congelabilidade do sêmen bovino / Seminal plasma proteins related to bovine semen freezabilty

A tecnologia da criopreservação tem apresentado desenvolvimento notório, tanto no melhoramento da qualidade como na fertilidade do sêmen após o processamento. Apesar dos progressos alcançados com as técnicas da criopreservação do sêmen, ainda são obtidas perdas em torno de 50% de espermatozóides viáveis durante o processo (Watson, 2000). Os indivíduos podem ser classificados como produtores de sêmen de alta e baixa congelabilidade, dependendo das características estruturais da membrana, o que é geneticamente determinado, predispondo à sobrevivência ao estresse da criopreservação (WATSON, 2000). Várias proteínas do plasma seminal bovino têm influência na congelabilidade do sêmen. Roncoletta (1999), através da análise das proteínas do plasma seminal, verificaram três polipepteptídeos (45, 24 e 10,8 kDa) com densidade óptica superior nas amostras de reprodutores de melhor congelabilidade do sêmen. Outras proteínas também foram relacionadas à alta congelabilidade do sêmen: a proteína ácida do fluído seminal bovino (aSFP), a clusterina, a albumina e a osteopontina (OPN); podendo assim, ser indicadas como marcadores. Também foram indicadas mais duas bandas protéicas (15-16 kDa, pI 4,7-5,2 e relacionadas à alta congelabilidade do sêmen, porém que não foram identificadas no estudo, mas que pelo peso molecular e ponto isoelétrico aproximado poderiam corresponder às proteínas BSP A1/A2 (15-16 kDa, pI 4,7-5,2) ou BSP A3 (13-14 kDa, pI 6,0-6,5) (JOBIM et al., 2004). Outros estudos indicam ainda as proteínas P25b e as proteínas anticongelação (AFPs), como proteínas relacionadas a congelabilidade do sêmen. Apesar dos progressos no conhecimento das proteínas relacionadas à criopreservação, ainda encontramos resultados bastante contraditórios e por vezes, a ausência do conhecimento da forma de proteção conferida por determinadas proteínas... (AU)

Cryopreservation techniques has shown remarkable development, both in improving the quality and the fertility of semen after processing. Despite these progress , losses are around 50% of viable sperm during the process (Watson, 2000). Individuals can be classified as producers of high-and low semen The structural characteristics of the membrane, which is genetically determined, predisposes the survival of the stress of cryopreservation (Watson, 2000). Several proteins of bovine seminal plasma influence on semen freezability . Roncoletta (1999), analysing seminal plasma proteins, find three polipepteptídeos (45, 24 and 10.8 kDa) with higher optical density in samples of the bulls with high freezability semen. Other proteins were also highly related to the frozen semen: the acid bovine seminal fluid (aSFP), the clusterin, the albumin and osteopontin (OPN) can be shown as semen freezability markers. Also two protein bands, (15-16 kDa, pI 4,7-5,2) are related to the high semen freezability, based in molecular weight and isoelectric point could correspond to the protein BSP A1/A2 (15-16 kDa, pI 4,7-5,2) or BSP A3 (13-14 kDa, pI 6,0-6,5) (JOBIM, et al 2004). Other studies also indicate the proteins P25b as antifreezeprotein (AFPs) related to semen freezability... (AU)

Proteínas do plasma seminal relacionadas a congelabilidade do sêmen bovino / Seminal plasma proteins related to bovine semen freezabilty

A tecnologia da criopreservação tem apresentado desenvolvimento notório, tanto no melhoramento da qualidade como na fertilidade do sêmen após o processamento. Apesar dos progressos alcançados com as técnicas da criopreservação do sêmen, ainda são obtidas perdas em torno de 50% de espermatozóides viáveis durante o processo (Watson, 2000). Os indivíduos podem ser classificados como produtores de sêmen de alta e baixa congelabilidade, dependendo das características estruturais da membrana, o que é geneticamente determinado, predispondo à sobrevivência ao estresse da criopreservação (WATSON, 2000). Várias proteínas do plasma seminal bovino têm influência na congelabilidade do sêmen. Roncoletta (1999), através da análise das proteínas do plasma seminal, verificaram três polipepteptídeos (45, 24 e 10,8 kDa) com densidade óptica superior nas amostras de reprodutores de melhor congelabilidade do sêmen. Outras proteínas também foram relacionadas à alta congelabilidade do sêmen: a proteína ácida do fluído seminal bovino (aSFP), a clusterina, a albumina e a osteopontina (OPN); podendo assim, ser indicadas como marcadores. Também foram indicadas mais duas bandas protéicas (15-16 kDa, pI 4,7-5,2 e relacionadas à alta congelabilidade do sêmen, porém que não foram identificadas no estudo, mas que pelo peso molecular e ponto isoelétrico aproximado poderiam corresponder às proteínas BSP A1/A2 (15-16 kDa, pI 4,7-5,2) ou BSP A3 (13-14 kDa, pI 6,0-6,5) (JOBIM et al., 2004). Outros estudos indicam ainda as proteínas P25b e as proteínas anticongelação (AFPs), como proteínas relacionadas a congelabilidade do sêmen. Apesar dos progressos no conhecimento das proteínas relacionadas à criopreservação, ainda encontramos resultados bastante contraditórios e por vezes, a ausência do conhecimento da forma de proteção conferida por determinadas proteínas...

Cryopreservation techniques has shown remarkable development, both in improving the quality and the fertility of semen after processing. Despite these progress , losses are around 50% of viable sperm during the process (Watson, 2000). Individuals can be classified as producers of high-and low semen The structural characteristics of the membrane, which is genetically determined, predisposes the survival of the stress of cryopreservation (Watson, 2000). Several proteins of bovine seminal plasma influence on semen freezability . Roncoletta (1999), analysing seminal plasma proteins, find three polipepteptídeos (45, 24 and 10.8 kDa) with higher optical density in samples of the bulls with high freezability semen. Other proteins were also highly related to the frozen semen: the acid bovine seminal fluid (aSFP), the clusterin, the albumin and osteopontin (OPN) can be shown as semen freezability markers. Also two protein bands, (15-16 kDa, pI 4,7-5,2) are related to the high semen freezability, based in molecular weight and isoelectric point could correspond to the protein BSP A1/A2 (15-16 kDa, pI 4,7-5,2) or BSP A3 (13-14 kDa, pI 6,0-6,5) (JOBIM, et al 2004). Other studies also indicate the proteins P25b as antifreezeprotein (AFPs) related to semen freezability...

Transferrin and albumin polimorphism and its correlation with sexual maturity in bulls / Polimorfismo da transferrina e albumina e as associações com precocidade sexual em bovinos da raça Nelore doadores de sêmen

The association between biochemical polymorphic systems and early sexual maturity was assessed subjecting 16 animals (from 17 to 36 months old) to eletrophoretic studies, searching for transferrin and albumin polymorphisms. Two albumin alleles, AlbA (AlbF) and AlbB (AlbS), but only either as AlbB homozigotes or AlbAB heterozigotes were observed. There were no AlbA homozigotes. It was not possible to detect relationships between albumin genotypes and early sexual maturity. Regarding transferrin, it was possible to detect two alleles (TfD and TfE). When trying to establish a relationship between transferrin electrophoretic pattern and the desirable economic trait (early sexual maturity) it is suggested that the TfD allele could be associated with bulls that probably will provide freezable semen at an early age.

O presente estudo utilizou 16 animais Bos taurus indicus da raça Nelore doadores de sêmen. Estes animais foram divididos em grupos de acordo com a idade em que o sêmen congelou pela primeira vez. O grupo I, considerado precoce, apresentou animais com sêmen passível de congelação com idade inferior a 20 meses. O grupo II, composto por animais que tiveram o sêmen congelado com idade entre 21 e 26 meses. E o grupo III, tido como tardio, composto por animais com sêmen congelável com idade superior a 27 meses. Para análise dos padrões eletroforéticos da transferrina e albumina, amostras de sangue foram colhidas em tubos heparinizados e submetidos a centrifugação de 2.500 G por 15 minutos para separação do plasma sangüíneo. As amostras de plasma sangüíneo foram processadas para que a corrida eletroforética em gel de poliacrilamida pudesse ser realizada. Para a coloração do gel, usou-se Coomasie Brilliant Blue. Após análise dos padrões eletroforéticos da transferrina e albumina, observou-se que não houve relação detectável entre os fenótipos da albumina e precocidade sexual de touros doadores. Entretanto, em relação à transferrina, foi possível sugerir uma associação entre o alelo TfD com touros portadores de sêmen congelável precocemente ou medianamente em termos de idade à congelação.