Utilisation of sperm-binding assay combined with computer-assisted sperm analysis to evaluate frozen-thawed bull semen.

Due to homologies between the chicken egg perivitelline membrane with mammalian zona pellucida proteins, spermatozoa of several species are able to bind to this membrane. However, adequate standardisation is required to attest possible applications of this technique for semen evaluation of a given species. Therefore, we thawed and divided cryopreserved semen samples into two aliquotes, one kept in water bath at 37 °C (thawed) and the other submitted to snap-freezing to damage sperm cells (dead spermatozoa). Aliquotes were mixed into different ratios of thawed:dead cells and analysed for motility, membrane and acrosomal integrity, and mitochondrial activity. In parallel, chicken egg perivitelline membranes were inseminated with these ratios, and the number of spermatozoa bound per mm(2) of membrane was assessed by conventional microscopy (CM) and computer-assisted sperm analysis (CASA). Linear regression showed high correlation between thawed:dead sperm ratio and number of spermatozoa bound to the membrane (CM: r(2) = 0.91 and CASA: r(2) = 0.92 respectively). Additionally, positive correlations were found between the number of spermatozoa bound to the membrane and acrosomal integrity, membrane integrity, mitochondrial activity and motility. These findings indicate that sperm-egg-binding assay associated with CASA is a reliable, practical and inexpensive method for examining the fertilising capacity of cryopreserved bull semen.

Seasonal variation in semen quality in Bos indicus and Bos taurus bulls raised under tropical conditions.

In the present study, we tested the hypothesis that Bos taurus taurus bulls have greater reactive oxygen species (ROS) and lower activity of antioxidant enzymes in their semen than Bos taurus indicus bulls. Sixteen Simmental bulls (B. t. taurus) and 11 Nelore bulls (B. t. indicus) were managed extensively in a tropical environment. Semen was collected twice annually (summer and winter) for 2 consecutive years. Simmental bulls had significantly higher percentages of major sperm defects during the summer than the winter (20.3+/-3.1% versus 12.2+/-2.4%, respectively; mean+/-S.E.M.). There was an interaction of breed and season for minor sperm defects (P=0.037; highest in Nelore bulls in the summer) and an effect of season on total defects (P=0.066; higher in summer). To evaluate oxidative damage, malondialdehyde (lipid-peroxidation metabolite) concentrations were indirectly measured by semen concentrations of thiobarbituric acid reactive substances (TBARS); these were higher in summer than in winter (728.1+/-79.3ng/mL versus 423.8+/-72.6ng/mL, respectively; P=0.01). Glutathione peroxidase/redutase (GPx) activity in semen was higher in Simmental versus Nelore bulls (741.6+/-62.1 versus 510.2+/-62.8; P<0.01). However, superoxide dismutase (SOD), another antioxidant enzyme, was not significantly affected by breed or season. There were correlations between TBARS and sperm primary defects during the summer for both Simmental and Nelore bulls (r=0.59, P=0.021 and r=0.40, P=0.034, respectively), and between SOD and primary defects during summer for Simmental bulls only (r=-0.51, P=0.041). In conclusion, there was a higher level of lipid peroxidation (ROS) in semen of Simmental versus Nelore bulls; apparently the higher GPx activity in Simmental bulls was insufficient to avoid damage that occurred concurrent with increased ROS production during the summer.

Effects of dexamethasone treatment (to mimic stress) and Vitamin E oral supplementation on the spermiogram and on seminal plasma spontaneous lipid peroxidation and antioxidant enzyme activities in dogs.

The objective was to determine if treatment with dexamethasone (to mimic stress) has a deleterious effect on the spermiogram and on the composition of seminal plasma in the dog and whether adverse effects were reduced by oral supplementation with Vitamin E. Eighteen adult male Rottweiler dogs were randomly allocated in a 2 x 2 factorial treatment design (with or without dexamethasone treatment versus with or without Vitamin E supplementation). Dogs in the supplemented group received 500 mg of alpha-tocopherol (Vitamin E)/dog/day per os for 10 weeks. Dexamethasone (0.01 mg/kg/day i.m.) was given once daily for 7 days, starting 7 days after the onset of Vitamin E supplementation. Food intake, body condition score and body weight were assessed daily. Semen collections (digital manipulation) were performed twice weekly for 14 weeks and blood samples (for plasma concentrations of cortisol and testosterone) were collected once a week. Dexamethasone treatment significantly reduced ejaculate volume and increased thiobarbituric acid-reactive substances (TBARS) in the seminal plasma. In contrast, supplementation with Vitamin E increased sperm motility, vigor and concentration and decreased the percentage of major sperm defects. In conclusion, dexamethasone treatment (to mimic stress) had a deleterious effect on the spermiogram and on the seminal plasma lipid peroxidation in dogs; however, some of these effects were prevented by oral supplementation with Vitamin E.

Plasma concentrations of estradiol 17beta and PGF2alpha metabolite and placental fatty acid composition and antioxidant enzyme activity in cows with and without retained fetal membranes.

Fetal membrane retention is one of the most common problems in Holstein cattle after parturition. To investigate mechanisms involved, the following parameters were studied in the peri-parturition period: plasmatic concentrations of estradiol-17beta (E2) and PGFM (PGF2alpha metabolite), activity of antioxidant enzymes (superoxide dismutase-SOD, catalase-CAT and glutathione peroxidase-GSH-Px), thiobarbituric acid reagent substances (TBAR) concentrations and fatty acid composition of the placentae. E2 at parturition in the NPR group (control cows, n = 10) was higher than in PR cows (placental retention, n = 10) (P < 0.05). Activity of SOD in fetal tissue of NPR animals was higher than that of the PR group. In contrast, there was no difference between the two groups in activity of GSH-Px and CAT and the TBAR content of placental tissues. PR maternal tissues had proportionally more arachidonic and linoleic acid than tissues from NPR cows. Therefore, a complex of sequential events may cause placenta retention, starting with an unbalance of antioxidant capacity of the placenta, followed by a decrease in production of estrogen, which leads to the accumulation of arachidonic and linoleic acid in placental tissues.

Is the thoroughbred race-horse under chronic stress?

Thoroughbred fillies were divided into three groups according to age: group 1, 7 fillies aged 1 to 2 years (G1) starting the training program; group 2, 9 fillies aged 2 to 3 years (G2) in a full training program; group 3, 8 older fillies 3 to 4 years of age (G3) training and racing. Blood samples were collected weekly from July to December. Cortisol was quantified using a solid phase DPC kit. The intra- and interassay coefficients of variation were 12.5% and 15.65% and sensitivity was 1.9 +/- 0.2 nmol/l. The semester average of cortisol levels varied between groups: G1 = 148.8 +/- 6.7, G2 = 125.7 +/- 5.8, G3 = 101.1 +/- 5.4 nmol/l, with G3 differing statistically from the other groups. The lower cortisol levels observed in the older fillies leads us to propose that the stress stimulus, when maintained over a long period of time, may become chronic and result in a reduction of hypophyseal corticotropin-releasing hormone receptors. The secretion of endogenous opioids may also lead to low serum cortisol levels.

Changes in the fecal concentrations of cortisol and androgen metabolites in captive male jaguars (Panthera onca) in response to stress.

In the present study we determined the efficacy of the measurement of fecal cortisol and androgen metabolite concentrations to monitor adrenal and testicular activity in the jaguar (Panthera onca). Three captive male jaguars were chemically restrained and electroejaculated once or twice within a period of two months. Fecal samples were collected daily for 5 days before and 5 days after the procedure and stored at -20 degrees C until extraction. Variations in the concentrations of cortisol and androgen metabolites before and after the procedure were determined by solid phase cortisol and testosterone radioimmunoassay and feces dry weight was determined by drying at 37 degrees C for 24 h under vacuum. On four occasions, fecal cortisol metabolite levels were elevated above baseline (307.8 +/- 17.5 ng/g dry feces) in the first fecal sample collected after the procedure (100 to 350% above baseline). On one occasion, we did not detect any variation. Mean (+/- SEM) fecal androgen concentration did not change after chemical restraint and electroejaculation (before: 131.1 +/- 26.7, after: 213.7 +/- 43.6 ng/g dry feces). These data show that determination of fecal cortisol and androgen metabolites can be very useful for a noninvasive assessment of animal well-being and as a complement to behavioral, physiological, and pathological studies. It can also be useful for the study of the relationship between adrenal activity and reproductive performance in the jaguar.

Pre-parturition profile of steroids and prostaglandin in cows with or without foetal membrane retention.

Retained foetal membranes in cattle is one of the most common complications associated to the reduction in milk yield and impaired fertility in dairy cattle. In order to determine some endocrine mechanisms controlling parturition and delivery of foetal membranes, plasma concentrations of steroids and prostanoids were determined in 20 healthy Holstein cows. Samples were taken within the interval of 5 days pre-parturition to 12h after calving. Progesterone (P4) levels were similar in cows with (PR) and without (NPR) placental retention. While the estradiol-17beta (E2) peak at parturition was lower in PR than in NPR cows, cortisol levels were greater in PR cows 12 and 24h pre-parturition. The Prostaglandin F2alpha metabolite (PGFM) levels were higher at parturition in NPR compared with the PR group, but 12h later, these levels in the PR group increased so that concentrations were greater as compared with NPR cows. The Prostaglandin E2 metabolite (PGEM), 24, 48 and 72 h pre-parturition, were higher in PR cows. However, the PGFM:PGEM ratio was greater in cows up NPR at all time when included, indicating the importance of higher levels of Prostaglandin F2alpha (PGF2alpha) than Prostaglandin E2 (PGE2) for normal placental delivery. In conclusion, placental retention was related to both estrogen and PGF2alpha deficiency, which may be a consequence of metabolic stress leading to PGE2 and maternal cortisol synthesis before parturition.

Puberty and growth rate in thoroughbred fillies.

We measured progesterone and estradiol levels from birth to the beginning of adult life in 10 Thoroughbred fillies from the Equilia Stud Farm in Avaré SP, Brasil. The animals were measured and weighed monthly for the determination of body development and of a possible correlation between the rate of weight and height gain and the onset of detectable sex hormone levels. Jugular blood was collected twice a week and stored at -20 degrees C until assay of progesterone by a solid phase RIA with a sensitivity of 0.32 nmol L and of estradiol by liquid phase RIA adapted to low levels (3.67 pmol L ). The fillies were born with high serum levels of both hormones, which fell to undetectable levels by the first week of life. A variation in growth rate was observed, with the highest levels occurring from birth to the 3rd month of life, followed by a reduction until 15 mo of life, when fast growth was resumed. The monthly weight gain was 1.5% when the fillies reached puberty and 5.4% during the next month, (P < 0.05, Friedman test). During this second period of accelerated growth after the beginning of progesterone production at detectable levels (above 0.318 nmol L ), the parameters of skeletal growth did not differ (P > 0.05). The month of onset of puberty was the month of lowest weight gain in the life of the fillies, and it coincided with the highest insolation period. In conclusion, horses, like all other developed vertebrates, have a double pattern of development, with the acceleration observed at puberty depending on sex steroids, which in turn coincides with the highest insolation period. Gonadal activity characterized by serum progesterone levels was low from birth to the onset of puberty. After puberty the progesterone cycles were similar to those of adult animals with a mature hypothalamic-gonadal axis.

Collection and evaluation of semen from captive howler monkeys (Alouatta caraya).

Semen samples (n=58) were collected by electroejaculation from nine adult male howler monkeys (Alouatta caraya) between November 2000 and August 2001 at the National Primates Center, Ananindeua, Brazil. The ejaculates were free of coagulum. Mean (+/-S.D.) values were: volume, 0.09 +/- 0.05 ml; pH, 8.1 +/- 0.5; concentration 649.5 +/- 926.7 x 10(6) sperm/ml; progressive motility, 75.8 +/- 18.1%; forward progressive sperm motility (scale, 0-5), 3.5 +/- 1.0; live spermatozoa, 68.3 +/- 15.0%; primary defects, 9.6 +/- 4.5%; and secondary defects, 11.8 +/- 4.6%. There were high correlations between motility and live sperm (r = 0.91, P < 0.01), motility and forward progressive sperm motility (r = 0.84, P < 0.01) and between forward progressive sperm motility and live sperm (r = 0.78, P < 0.01). There were no alterations observed during clinical examinations and hematological analysis performed before and after semen collection. Therefore, the method was considered safe and efficient. It can be used for the evaluation of the breeding potential of male howler monkeys in captivity and for the establishment of new assisted reproductive technology (ART) for threatened species of neotropical primates.

Evaluation of the Karras spermdensimeter in relation to the Neubauer counting chamber for sperm concentration measurement in boar semen.

The correct measurement of spermatic concentration in boar semen is one of the basic tasks routinely performed at the Swine Artificial Insemination Center (CIAS). This article explains two experiments whose objectives were: Experiment 1: This trial had two objectives: (1) to evaluate the precision of the spermdensimeter (SPM) with relation to the Neubauer count chamber (CN) which is considered the standard technique in the field and (2) to correct the scale on the SPM table. Experiment 2: This trial also had two objectives: (1) to evaluate the time and repetitions required in the SPM and (2) to compare the SPM adjusted table produced in Expt 1 with the SPM standard table. The methods in Expt 1 were used to measure total spermatic concentration in 141 boar ejaculates. The average spermatic concentrations (10(6) spermatozoids/ml) and average dose number were 229.1 and 22.6 for CN, and 283.6 and 28.0 for SPM. These results indicate that the SPM has a tendency to overestimate spermatic concentration with relation to the CN. The SPM produced a smaller coefficient of variation and needed lesser examination time than the CN (p < 0.05). The SPM adjusted table created in Expt 1 produced similar results as the SPM and CN standard tables (2.96 billion live spermatozoids/dose vs 2.36 billion live spermatozoids/dose). Incorporation and evaluation of supplemental data from other CIAS regarding the use of the SPM adjusted table in the field entitled 'Adjusted Table for Karras Spermdensimeter LPS-FMVZ-USP' would be valuable to prove its efficiency.

Detecção de estro em jaguatirica (Leopardus pardalis) utilizando citologia vaginal / Vaginal cytology for estrus detection in ocelot (Leopardus pardalis)

Utilizou-se a citologia vaginal por meio de diferentes métodos de coloração para detecção de cio em jaguatirica, Leopardus pardalis, pela estimulação hormonal exógena e pela avaliação das estruturas ovarianas por videolaparoscopia. Cinco fêmeas foram tratadas com eCG/hCG e FSH/LH a cada quatro meses pelo período de dois anos. Videolaparoscopia foi realizada após cada tratamento utilizando-se cetamina-xilazina e isoflurano. Esfregaços vaginais foram obtidos 15 dias antes e após a videolaparoscopia. As lâminas foram analisadas ao microscópio de luz quanto aos tipos celulares predominantes. Todos os animais apresentaram folículos maduros (>2mm) e corpos lúteos recentes em todas as intervenções. Não houve diferença significativa entre os resultados obtidos na mesma coloração de acordo com os tratamentos utilizados. Todas as técnicas mostraram-se eficientes na detecção de células superficiais queratinizadas anucleadas e nucleadas, intermediárias, parabasais e basais. Foi possível determinar a fase de estro em Leopardus pardalis por meio da citologia vaginal.

Vaginal cytology was evaluated for estrus detection using different stains after hormonal stimulation with exogenous gonadotrophin (eCG/hCG, FSH/LH) and videolaparoscopy for ovarian structure evaluation. Five L. pardalis were treated four times during two years. After each treatment, videolaparoscopy was performed using Ketamine-Xylazine and Isoflurane. Vaginal cytology was made 15 days before and after videolaparoscopy. Three stains were used: Diff Quick, Papanicolaou, and Shorr. The slides were analyzed for the typical cell predominance. All the animals showed mature follicles (>2mm) and recent corpus luteum in all procedures. No significative difference was observed between the results in the same stain according to the treatment eCG/hCG and FSH/LH. All stains were efficient in detection of nucleated and anuclear superficial keratinized cells; intermediated, parabasal, and basal cells. Vaginal cytology can be used for estrus detection in Leopardus pardalis.

Comparative analyses of semen and endocrine characteristics of free-living versus captive jaguars (Panthera onca).

Semen and blood samples were obtained from free-living (n = 6) and captive (n = 8) jaguars (Panthera onca) to compare reproductive characteristics between the two populations. Semen samples were analysed for volume (ml), percentage of motile spermatozoa, rate of forward progression (0-5), concentration (10(6) ml(-1)), total sperm count (10(6)) and sperm morphology. Serum testosterone concentration was determined by radioimmunoassay. Although ejaculate volume was greater in captive jaguars (n = 47 samples) than in free-living jaguars (n = 7 samples) (P < 0.05), the free-living jaguars produced more total spermatozoa (59.3 +/- 12.8 versus 152.0 +/- 88.0 x 10(6), respectively; not significant) with better viability and forward progression (2.8 +/- 0.1 versus 3.5 +/- 0.2, respectively; P < 0.05) and more spermatozoa with normal morphology (73.5 +/- 3.9 versus 5.0 +/- 1.1%, respectively; P < 0.05). Serum testosterone concentrations were similar for captive and free-living male jaguars (3.1 +/- 0.7 and 2.1 +/- 0.8 ng ml(-1), respectively). In summary, the data showed that semen may be collected successfully from free-living jaguars and evaluated under field conditions to establish normative reproductive values in this species. The results also indicate that jaguars maintained in zoos show inferior seminal characteristics compared with free-living animals.

Is the throughbred race-horse under chronic stress?

Thoroughbred fillies were divided into three groups according to age: group 1, 7 fillies aged 1 to 2 years (G1) starting the training program; group 2, 9 fillies aged 2 to 3 years (G2) in a full training program; group 3, 8 older fillies 3 to 4 years of age (G3) training and racing. Blood samples were collected weekly from July to December. Cortisol was quantified using a solid phase DPC kit. The intra- and interassay coefficients of variation were 12.5 percent and 15.65 percent and sensitivity was 1.9 + 0.2 nmol/1. The semester average of cortisol levels varied between groups: G1 = 148.8 + 6.7, G2 = 125.7 + 5.8, G3 = 101.1 + 5.44 nmol/l, with G3 differing statistically from the other groups. The lower cortisol levels observed in the older fillies leads us to propose that the stress stimulus, when maintained over a long period of time, may become chronic and result in a reduction of hypophyseal corticotropin-releasing hormone receptors. The secretion of endogenous opioids may also lead to low serum cortisol levels.

Changes in the fecal concentrations of cortisol and androgen metabolites in captive male jaguars (Panthera onca) in response to stress

In the present study we determined the efficacy of the measurement of fecal cortisol and androgen metabolite concentrations to monitor adrenal and testicular activity in the jaguar (Panthera onca). Three captive male jaguars were chemically restrained and electroejaculated once or twice within a period of two months. Fecal samples were collected daily for 5 days before and 5 days after the procedure and stored at -20ºC until extraction. Variations in the concentrations of cortisol and androgen metabolites before and after the procedure were determined by solid phase cortisol and testosterone radioimmunoassay and feces dry weight was determined by drying at 37ºC for 24 h under vacuum. On four occasions, fecal cortisol metabolite levels were elevated above baseline (307.8 ± 17.5 ng/g dry feces) in the first fecal sample collected after the procedure (100 to 350 percent above baseline). On one occasion, we did not detect any variation. Mean (± SEM) fecal androgen concentration did not change after chemical restraint and electroejaculation (before: 131.1 ± 26.7, after: 213.7 ± 43.6 ng/g dry feces). These data show that determination of fecal cortisol and androgen metabolites can be very useful for a noninvasive assessment of animal well-being and as a complement to behavioral, physiological, and pathological studies. It can also be useful for the study of the relationship between adrenal activity and reproductive performance in the jaguar.

Colheita e avaliaçäo do sêmen em onça pintada (Panthera onca) / Semen collection and evaluation in the jaguar (Panthera onca)

Foram submetidas à eletroejaculaçäo 10 onças pintadas, de origem desconhecida, mantidas na Fundaçäo Parque Zoológico de Säo Paulo (n = 7) e Parque Zoológico Municipal "Quinzinho de Barros"- Sorocaba (n = 3), recebendo alimentaçäo de acordo com a dieta de rotina de cada instituiçäo. Nos 54 procedimentos realizados, foram obtidos ejaculados, ocorrendo contaminaçäo por urina em 3,7 por cento (n = 2) e em 11 (20,4 por cento) ocasiöes em que näo foram observados espematozóides. O volume médio obtido foi 7,42 ñ 3,69 ml, com motilidade média de 62,6 ñ 11,0 por cento e vigor de 2,71 ñ 0,52. A concentraçäo espermática foi 6,20 ñ 3,03 x 10 elevado a sexta potência espematozóides/ml, sendo que a porcentagem média de espermatozóides morfologicamente normais foi 46,7 ñ 5,8 por cento. A técnica de colheita de sêmen por eletroejaculaçäo é eficiente na espécie Panthera onça

Efeitos do hormônio folículo estimulante (FSH) e da gonadotrofina da menopausa humana (hMG) como agentes superovulantes em tratamentos sucessivos de vacas da raça Holandesa variedade preta e branca, utilizadas em transferência de embriöes / Effect of follicle stimulating hormone (FSH) and human menopausae gonadotrophin (hMG) as superovulating agents in consecutive treatments of Holstein black and white cows used in embryo transfer

Para verificar o efeito superovulante de 38mg de FSH e de 2400 U.I. de hMG (1200 U.I. de FSH + 1200 U.I. de LH) em doses diárias decrescentes durante 4 dias consecutivos, a partir do 10§ dia do ciclo estral, foram utilizadas 10 vacas Holandêsas, variedade preta e branca. No 3§ dia após o início do tratamento, os animais receberam 500 ug de Cloprostenol para sincronizaçäo do ciclo estral, sendo inseminados 12 e 24 horas após o início do cio. O processo de superovulaçäo foi repetido por 5 vezes, com intervalo de 60 dias, empregando-se os mesmos animais e as mesmas doses hormonais para verificar as possíveis alteraçöes das respostas superovulatórias. Realizaram-se as colheitas dos embriöes no 7§ dia após o cio, através do método näo cirúrgico, em sistema fechado e com o auxílio de catéter Neustadt Ad-Aisch. Utilizaram-se, para cada corno uterino, 500ml do meio de Dulbecco modificado (PBS), aquecidos a 37§C e enriquecidos com 1 por cento de soro fetal bovino. O meio recuperado permaneceu em repouso por 30 minutos, sifonando-se o sobrenadante. Os 100ml restantes foram colocados em placas de Petri quadriculadas para localizaçäo das estruturas, empregando-se estereomicroscópio com aumentos de 10 e 40 vezes. Das 105 estruturas obtidas com FSH e 43 com hMG, 79 (75 por cento) e 31 (72 por cento) eram viáveis, sendo os embriöes inovulados nos cornos uterinos ipsolaterais aos corpos lúteos através do método näo cirúrgico. O diagnóstico de gestaçäo foi realizado através de palpaçäo retal, 45-60 dias após as transferências, obtendo-se taxas de 43 por cento (34/79) para o FSH e 55 por cento (17/31) para o hMG

Electroejaculation and seminal characteristics in chinchilla (Chinchilla laniger)

O método da eletroejaculaçäo foi utilizado em chinchila através de eletrodo introduzido a 3 cm no reto do animal, obtendo-se os melhores resultados com uma série de 9 choques de 12,5 mA, 6 choques de25,0 mA e 19 choques de 50,0 mA. O sêmen obtido nestas condiçöes foi considerado de boa qualidade, comportando-se satisfatoriamente tanto no processo de congelamento, quanto no descongelamento

Características seminais de cäes da raça Pastor Alemäo / Seminal characteristics of German Shepherd dogs

Sêmen de 25 cäes da raça Pastor Alemäo, com 1 a 7 anos de idade e pesando 30 a 35 quilos, foi coletado por masturbaçäo. Com o objetivo de utilizar o material para inseminaçäo artificial, colheu-se a segunda e parte da terceira fraçäo, constatando-se, em média, volume de 7,17 ml; cor branca e aspecto leitoso; motilidade de 68,84 por cento; vigor de 3,5; concentraçäo de 136.192 espermatozóides por mm3 e de 889.772.000 por ejaculado; defeitos espermáticos maiores de 9,92 por cento e menores de 7,62 por cento. O pH do sêmen, dos diluidores e do sêmen diluído variou, respectivamente, de 6,02 a 6,60; 5,85 a 6,90; e 5,97 a 6,76; a pressäo osmótica de 285,20 a 295,00; 240,00 a 310,00; e 272,32 a 303,92 mOsmois e a concentraçäo de sódio e potássio, respectivamente, para o plasma seminal e os 4 diluidores, de 134,40 a 156,00; 7,80 a 13,72; 3,00 a 270,00; e 6,00 a 25,00 mEq/l. Verificou-se que entre cäes, houve variaçäo significante de todas as características seminais, exceto a pressäo osmótica e, ainda, correlaçöes entre os caracteres seminais e entre estes e os totais de patologias espermáticas. A variabilidade das características seminais entre os cäes foi bem maior quando comparada entre as colheitas de cada animal, exceto a pressäo osmótica e a concentraçäo de sódio, que se apresentaram de maneira inversa