Helminth Fauna of Juvenile Green Sea Turtles (Chelonia mydas) from Rio de Janeiro State, Brazil.

The helminth fauna of juvenile green sea turtles (Chelonia mydas Linnaeus, 1758) is still poorly known. Herein, we study the gastrointestinal helminths of 28 juvenile green sea turtles found stranded on the north coast of Rio de Janeiro state, Brazil. All turtles were infected showing a rich helminth fauna. In total, 14802 trematodes belonging to 30 species and 5 families including Micros-caphidiidae, Plagiorchiidae, Pronocephalidae, Hapalotrematidae, and Telorchiidae were recovered. An unidentified nematode specimens was also found. The mean intensity was 536 (95% CI = 362 - 853) (range: 1 - 2831), and the species richness was 7.86 (95% CI = 6.46 - 9.21) (range: 1 - 17). The coast of Rio de Janeiro state represents new locality records for Angiodictyum posterovitellatum, Microscaphidium aberrans, M. warui, Octangium hyphalum, O. sagitta, Enodiotrema reductum and Pleurogonius laterouterus. This study confirms that the green sea turtle harbors the richest helminth fauna among sea turtle species and provides useful information on the gastrointestinal helminths of a poorly known stage in the life cycle of this endangered chelonian.

Ocular spirorchiidiosis in sea turtles from Brazil.

The causes of the beaching and death of sea turtles have not been fully clarified and continue to be studied. Mild, moderate and severe lesions caused by spirorchiidiosis have been seen for decades in different organs and were recently defined as the cause of death of a loggerhead turtle. In the present study, eyes and optic nerves were analysed in green sea turtles with spirorchiidiosis and no other debilitating factors. Injuries to the optic nerve and choroid layer were described in 235 animals (90%) infected with spirorchiids. Turtles with ocular spirorchiidiosis are approximately three times more likely to be cachectic than turtles with spirorchiidiosis without ocular involvement.

Zootechnical indices and digestibility in juveniles of tambaqui Colossoma macropomum fed a diet containing particulate maize.

Three experiments were done with different particle sizes of corn feed on its zootechnical performance, passing rate and apparent digestibility of juvenile tambaqui (Colossoma macropomum). In the first, 200 juveniles were used and distributed in 20 tanks (220 L), 10 fish per unit (four replicates). The experimental system used to record passage time was composed of five incubators with 200 L. In the second, 75 juveniles were used per 55 days (three replications). In the third, 75 juveniles were used and distributed in five incubators of 200 L. All experiments were performed randomly. Different linear behavior treatments were observed for apparent digestibility of crude protein; and the smaller particle size (150 µm) had better results digestibility. smaller particle size of the corn had better results, affected the growth performance of tambaqui and the apparent digestibility of crude protein and ether extract. Thus, is recommended that a particle size of 150 µm of corn be used for tambaqui.

Zootechnical indices and digestibility in juveniles of tambaqui Colossoma macropomum fed a diet containing particulate maize / Índices zootécnicos e digestibilidade em juvenis de tambaqui Colossoma macropomum alimentados com dieta contendo milho particulado

Three experiments were done with different particle sizes of corn feed on its zootechnical performance, passing rate and apparent digestibility of juvenile tambaqui (Colossoma macropomum). In the first, 200 juveniles were used and distributed in 20 tanks (220 L), 10 fish per unit (four replicates). The experimental system used to record passage time was composed of five incubators with 200 L. In the second, 75 juveniles were used per 55 days (three replications). In the third, 75 juveniles were used and distributed in five incubators of 200 L. All experiments were performed randomly. Different linear behavior treatments were observed for apparent digestibility of crude protein; and the smaller particle size (150 μm) had better results digestibility. Smaller particle size of the corn had better results, affected the growth performance of tambaqui and the apparent digestibility of crude protein and ether extract. Thus, is recommended that a particle size of 150 μm of corn be used for tambaqui.

Três experimentos foram realizados com diferentes tamanhos de partícula de ração para o desempenho zootécnico, taxa de aprovação e digestibilidade aparente de tambaqui juvenil (Colossoma macropomum). No primeiro, foram utilizados 200 juvenis e distribuídos em 20 tanques (220 L), 10 peixes por unidade (quatro repetições). O sistema experimental utilizado para registrar o tempo de passagem foi composto por cinco incubadoras com 200 L. No segundo, 75 juvenis foram utilizados por 55 dias (três repetições). No terceiro, 75 juvenis foram utilizados e distribuídos em cinco incubadoras de 200 L. Todos os experimentos foram realizados aleatoriamente. Diferentes tratamentos de comportamento linear foram observados para digestibilidade aparente da proteína bruta; e o menor tamanho de partícula (150 μm) apresentou melhor digestibilidade dos resultados. menor tamanho de partícula do milho apresentou melhores resultados, afetou o desempenho de crescimento de tambaquis e a digestibilidade aparente da proteína bruta e extrato etéreo. Portanto, recomenda-se o uso de um tamanho de partícula de 150 μm de milho para o tambaqui.

Zootechnical indices and digestibility in juveniles of tambaqui Colossoma macropomum fed a diet containing particulate maize

Abstract Three experiments were done with different particle sizes of corn feed on its zootechnical performance, passing rate and apparent digestibility of juvenile tambaqui (Colossoma macropomum). In the first, 200 juveniles were used and distributed in 20 tanks (220 L), 10 fish per unit (four replicates). The experimental system used to record passage time was composed of five incubators with 200 L. In the second, 75 juveniles were used per 55 days (three replications). In the third, 75 juveniles were used and distributed in five incubators of 200 L. All experiments were performed randomly. Different linear behavior treatments were observed for apparent digestibility of crude protein; and the smaller particle size (150 m) had better results digestibility. smaller particle size of the corn had better results, affected the growth performance of tambaqui and the apparent digestibility of crude protein and ether extract. Thus, is recommended that a particle size of 150 m of corn be used for tambaqui.

Resumo Três experimentos foram realizados com diferentes tamanhos de partícula de ração para o desempenho zootécnico, taxa de aprovação e digestibilidade aparente de tambaqui juvenil (Colossoma macropomum). No primeiro, foram utilizados 200 juvenis e distribuídos em 20 tanques (220 L), 10 peixes por unidade (quatro repetições). O sistema experimental utilizado para registrar o tempo de passagem foi composto por cinco incubadoras com 200 L. No segundo, 75 juvenis foram utilizados por 55 dias (três repetições). No terceiro, 75 juvenis foram utilizados e distribuídos em cinco incubadoras de 200 L. Todos os experimentos foram realizados aleatoriamente. Diferentes tratamentos de comportamento linear foram observados para digestibilidade aparente da proteína bruta; e o menor tamanho de partícula (150 m) apresentou melhor digestibilidade dos resultados. menor tamanho de partícula do milho apresentou melhores resultados, afetou o desempenho de crescimento de tambaquis e a digestibilidade aparente da proteína bruta e extrato etéreo. Portanto, recomenda-se o uso de um tamanho de partícula de 150 m de milho para o tambaqui.

Zootechnical indices and digestibility in juveniles of tambaqui Colossoma macropomum fed a diet containing particulate maize / Índices zootécnicos e digestibilidade em juvenis de tambaqui Colossoma macropomum alimentados com dieta contendo milho particulado

Three experiments were done with different particle sizes of corn feed on its zootechnical performance, passing rate and apparent digestibility of juvenile tambaqui (Colossoma macropomum). In the first, 200 juveniles were used and distributed in 20 tanks (220 L), 10 fish per unit (four replicates). The experimental system used to record passage time was composed of five incubators with 200 L. In the second, 75 juveniles were used per 55 days (three replications). In the third, 75 juveniles were used and distributed in five incubators of 200 L. All experiments were performed randomly. Different linear behavior treatments were observed for apparent digestibility of crude protein; and the smaller particle size (150 µm) had better results digestibility. smaller particle size of the corn had better results, affected the growth performance of tambaqui and the apparent digestibility of crude protein and ether extract. Thus, is recommended that a particle size of 150 µm of corn be used for tambaqui.

Três experimentos foram realizados com diferentes tamanhos de partícula de ração para o desempenho zootécnico, taxa de aprovação e digestibilidade aparente de tambaqui juvenil (Colossoma macropomum). No primeiro, foram utilizados 200 juvenis e distribuídos em 20 tanques (220 L), 10 peixes por unidade (quatro repetições). O sistema experimental utilizado para registrar o tempo de passagem foi composto por cinco incubadoras com 200 L. No segundo, 75 juvenis foram utilizados por 55 dias (três repetições). No terceiro, 75 juvenis foram utilizados e distribuídos em cinco incubadoras de 200 L. Todos os experimentos foram realizados aleatoriamente. Diferentes tratamentos de comportamento linear foram observados para digestibilidade aparente da proteína bruta; e o menor tamanho de partícula (150 µm) apresentou melhor digestibilidade dos resultados. menor tamanho de partícula do milho apresentou melhores resultados, afetou o desempenho de crescimento de tambaquis e a digestibilidade aparente da proteína bruta e extrato etéreo. Portanto, recomenda-se o uso de um tamanho de partícula de 150 µm de milho para o tambaqui.

Improvement of gamete quality and its short-term storage: an approach for biotechnology in laboratory fish.

In fish, in vitro fertilization is an important reproductive tool used as first step for application of others biotechniques as chromosome and embryo manipulation. In this study, we aimed to optimize gamete quality and their short-term storage from the yellowtail tetra Astyanax altiparanae, for future application in laboratory studies. Working with sperm, we evaluated the effects of spawning inducers (carp pituitary gland and Ovopel® [(D-Ala6, Pro9-NEt) - mGnRH+metoclopramide]) and the presence of female on sperm motility. Additionally, we developed new procedures for short-term storage of sperm and oocytes. Briefly, sperm motility was higher when male fish were treated with carp pituitary gland (73.1 ± 4.0%) or Ovopel® (79.5 ± 5.5%) when compared with the control group treated with 0.9% NaCl (55.6 ± 27.2%; P=0.1598). Maintenance of male fish with an ovulating female fish also improved sperm motility (74.4 ± 7.4%) when compared with untreated male fish (42.1 ± 26.1%; P=0.0018). Storage of sperm was optimized in modified Ringer solution, in which the sperm was kept motile for 18 days at 2.5°C. The addition of antibiotics or oxygen decreased sperm motility, but partial change of supernatant and the combination of those conditions improve storage ability of sperm. Fertilization ability of oocytes decreased significantly after storage for 30, 60 90 and 120 min at 5, 10, 15 and 20°C when compared with fresh oocytes (P=0.0471), but considering only the stored samples, the optimum temperature was 15°C. Those data describe new approaches to improve semen quality and gametes short-term storage in yellowtail tetra A. altiparanae and open new possibilities in vitro fertilization.

Transmembrane segment 10 is important for substrate recognition in Ga12 and Hxt2 sugar transporters in the yeast Saccharomyces cerevisiae.

A systematic series of chimeras between Ga12 galactose transporter and Hxt2 glucose transporter in yeast was produced to delineate the essential domain for substrate recognition. A domain of 101 amino acids close to the COOH-terminus that has been previously identified as the critical substrate recognition region was further divided into four subdomains, by introducing five restriction enzyme sites at exactly corresponding locations of both genes without changing coding amino acids. When each of all possible 16 modified genes was expressed, all the galactose transport-active chimeras were found to possess Ga12-derived transmembrane segment (TM) 10. Of the 35 amino acids in the TM1O region, only 12 differ between Ga12 and Hxt2, indicating that these 12 amino acids include the critical residue(s) responsible for the differential recognition of galactose and glucose in these transporters.

Histopathological findings in lungs of hawksbill turtles collected on the coasts of the states of Espírito Santo and Rio de Janeiro, Brazil / Achados histopatológicos em pulmões de tartarugas-de-pente procedentes dos litorais dos estados do Espírito Santo e do Rio de Janeiro, Brasil

The present work aimed to report the histopathological findings verified in lungs of hawksbill turtles (Eretmochelys imbricata) retrieved from the coasts of the states of Espírito Santo and Rio de Janeiro, Brazil. Between the years 2010 and 2014, 29 E. imbricata individuals were found stranded on the coasts, already dead or dying during treatment. Lung samples of all specimens were collected during necropsies, fixed in 10% neutral buffered formalin, subjected to routine histological processing and classified histomorphologically. The findings revealed that 37.93% of the specimens presented lung lesions. Of these, 90.91% were rescued alive and 9.09% were found dead; 63.63% were females and 36.37% were males. The mean weight was 5.44 Kg and the mean length 39 cm, characterizing young individuals. The animals originated from São Francisco de Itabapoana - RJ, Aracruz - ES, São Mateus - ES, Guarapari - ES, Linhares - ES, Itapemirim - ES, and Anchieta - ES. Macroscopic analysis revealed presence of foam, hyperemia, nodules in the parenchyma, cyst and caseous material. Microscopic examination evidenced heterophilic bronchopneumonia, parasitic granulomatous pneumonia caused by spirorchiids, bacterial granulomatous pneumonia, fungal granulomatous pneumonia, and congestion. It was concluded that juvenile specimens of Eretmochelys imbricata, females and males, originated from the states of Espírito Santo and Rio de Janeiro and found stranded both alive or dead, have significant lung lesions, mainly inflammatory ones, associated or not with infectious agents.(AU)

Objetivou-se com este trabalho relatar os achados histopatológicos em pulmões de Eretmochelys imbricata provenientes dos litorais dos estados do Espírito Santo e do Rio de Janeiro, Brasil. Amostras de pulmões de 29 E. imbricata foram coletadas, durante os anos de 2010 a 2014, em necropsias de espécimes encalhadas mortas ou que vieram a óbito durante o tratamento, nos litorais dos estados do Espírito Santo e do Rio de Janeiro, fixadas em formalina tamponada neutra a 10%, submetidas ao processamento histológico de rotina e classificadas histomorfologicamente. Os dados dos animais mostraram que 37,93% dos animais apresentaram lesões pulmonares. Destes, 90,91% foram encontrados vivos e 9,09% morto, 63,63% eram fêmeas e 36,37% machos. O peso médio foi de 5,44kg, e o comprimento médio de 39cm, o que caracterizou animais juvenis. Os animais eram provenientes de São Francisco de Itabapoana - RJ, Aracruz - ES, São Mateus - ES, Guarapari - ES, Linhares - ES, Itapemirim - ES e Anchieta - ES. Á macroscopia, observou-se: presença de espuma, hiperemia, nódulos no parênquima, cisto e cáseo. Ao exame microscópico, foram encontrados broncopneumonia heterofílica, pneumonia granulomatosa parasitária, causadas por espirorquídeos, pneumonia granulomatosa bacteriana, pneumonia granulomatosa fúngica e congestão. Conclui-se que exemplares juvenis de Eretmochelys imbricata, fêmeas e machos, provenientes dos estados do Espírito Santo e do Rio de Janeiro e encalhados tanto vivos quanto mortos, apresentam lesões pulmonares importantes, principalmente as de origem inflamatória, associadas ou não a agentes infecciosos.(AU)

Análise histomorfométrica do útero na espécie canina do nascimento aos seis meses de idade / Histomorphometric analysis of the uterus in dogs from birth to six months of age

Objetivou-se caracterizar para a espécie canina o desenvolvimento histológico uterino do nascimento aos seis meses de idade. Foram analisados úteros (n=32) de animais com idades entre um e 180 dias pós-nascimento (DPN), distribuídos em oito grupos: G1-1 DPN (1 Yorkshire Terrier, 1 Poodle, 2 Sem Raça Definida (SRD)/peso médio (Pm)=190g), G2-15 DPN (2 Yorkshire Terrier e 2 SRD/Pm=354g), G3-30 DPN (1 Rottweiler, 1 Poodle, 2 SRD/Pm=985g), G4-45 DPN (1 Poodle, 3 SRD/Pm=1,1kg), G5-60 DPN (1 Yorkshire Terrier, 1 Poodle e 2 SRD/Pm=1,4kg), G6-90 DPN (4 SRD/Pm=2,8kg), G7-120 DPN (1 Poodle e 3 SRD/Pm=6,6kg) e G8-180 DPN (1 Rottweiler, 1 Poodle e 2 SRD/Pm=11kg). A análise histológica constituiu de mensurações das espessuras (µm) da parede uterina, miométrio, endométrio, assim como diâmetro (µm) e número de glândulas endometriais. A análise estatística demonstrou estabilidade e homogeneidade nas estruturas avaliadas com coeficientes de variação baixos (<10%). Observou-se: útero com um DPN: epitélio com células cúbicas simples, miométrio rudimentar, presença de perimétrio e ausência de glândulas endometriais; aos 15 DPN: desenvolvimento de estrutura primordial de formação das glândulas endometriais; dos 30 aos 45 DPN: endométrio e glândulas endometriais simples; aos 60 DPN: glândulas endometriais em lâmina própria com ramificações e discreto pregueamento endometrial; de 90 a 180 DPN: todas as estruturas uterinas apresentaram histoarquitetura de um animal maduro. Todas as variáveis analisadas apresentaram correlação positiva com a idade pós-nascimento (R2≥72,2%). Conclui-se que o útero da cadela desenvolve-se continuamente do nascimento à 180 dias de vida e que apresenta-se desprovido de glândulas endometriais ao nascimento. As primeiras glândulas endometriais são observadas aos 15 dias de vida e apresenta conformação histológica de um animal adulto após 60 dias do nascimento.

This study aimed to characterize canine species' uterine histological development from birth to six months of age. Uteri (n = 32) of animals aged between one and 180 days postnatal (PND), distributed into eight groups were analyzed: G1-1 PND (1 Yorkshire Terrier, Poodle 1, 2 Mixed Breed (MB)/Medium weight (Mw) = 190g), G2-15 PND (2 Yorkshire Terrier and 2 MB/Mw = 354g), G3-30 PND (1 Rottweiler, 1 Poodle, 2 MB/Mw = 985g), G4-45 PND (1 Poodle, 3 MB/Mw = 1.1kg), G5-60 PND (1 Yorkshire Terrier, Poodle 1 and 2 MB/Mw = 1.4kg), G6-90 PND (4 MB/Mw = 2.8kg), G7-120 PND (1 Poodle and 3 MB/Mw = 6.6kg) and G8-180 PND (1 Rottweiler, Poodle 1 and 2 MB/Mw = 11kg). Histological examination consisted of thickness measurement (µm) of the uterine wall, myometrium, endometrium, as well as diameter (µm) and number of endometrial glands. Statistical analysis showed stability and uniformity in the evaluated structures with low coefficients of variation (<10%). We observed: uterus with one PND: simple cubic epithelium cells, rudimentary myometrium, perimetrium presence and absence of endometrial glands; at 15 PND: development of primordial structure formation of endometrial glands; from 30 to 45 PND: simple endometrium and endometrial glands; PND 60: endometrial glands in the lamina propria with branches and discreet endometrial pleating; 90-180 PND: uterine all structures presented histoarchitecture of a mature animal. All variables were positively correlated with postnatal age (R2 ≥ 72.2%). It is concluded that the uterus of the bitch continuously evolves from birth to 180 days old and is presented devoid of endometrial glands at birth. The first endometrial glands are observed at 15 days of life and present histological conformation to an adult 60 days after birth.

Substrate recognition domain of the Gal2 galactose transporter in yeast Saccharomyces cerevisiae as revealed by chimeric galactose-glucose transporters.

The Gal2 galactose transporter takes up galactose in yeast. A homologous glucose transporter from the same organism, Hxt2, was selected, and various chimeras between these two transporters were constructed by making use of homologous recombination in Escherichia coli. Comparison of the galactose transport activities of three series of chimeras enabled us to positively identify a crucial substrate recognition region of 101 amino acids that lies close to the carboxyl terminus of the Gal2 transporter.

Amino acid residues responsible for galactose recognition in yeast Gal2 transporter.

A novel, systematic approach was used to identify amino acid residues responsible for substrate recognition in the transmembrane 10 region of the Gal2 galactose transporter of Saccharomyces cerevisiae. A mixture of approximately 25,000 distinct plasmids that encode all the combinations of 12 amino acids in transmembrane 10 that are different in Gal2 and the homologous glucose transporter Hxt2 was synthesized. Selection of galactose transport-positive clones on galactose limited agar plates yielded 19 clones, all of which contained the Tyr446 residue found in Gal2. 14 of the 19 clones contained Trp455 found in Gal2, whereas the other 5 contained Cys455, a residue not found in either Gal2 or Hxt2. When Tyr446 of Gal2 was replaced with any of the other 19 amino acids, no galactose transport activity was observed in the resulting transporters, indicating that Tyr446 plays an essential role in the transport of this sugar. Replacement of 2 amino acids of Hxt2 with the corresponding Tyr446 and Trp455 of Gal2 allowed the modified Hxt2 to transport galactose. The Km of galactose transport for the modified transporter was 8-fold higher than that of Gal2. These results and other evidence unequivocally show that Tyr446 is essential and Trp455 is important for the discrimination of galactose versus glucose.

Nanosecond fluorescence studies of noncovalent interaction of monomeric and dimeric intercalators with DNA.

The noncovalent interaction of 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2) and its derivatives, which are potent mutagens isolated from L-glutamic acid pyrolysate, with calf thymus DNA was studied by steady-state and nanosecond fluorescence spectroscopies. The fluorescence of these compounds exhibits static quenching by noncovalent interaction with DNA. Fluorescence lifetimes of the free and intercalated states of these compounds were determined to be 9-10 and 0.5-1 ns, respectively. The bisintercalative effect of the dimeric analogue of Glu-P-2, bis(Glu-P-2)spermine (2GP-SP), to DNA was also investigated. This 2GP-SP, which has two Glu-P-2 moieties at each end of spermine, indicates a strong intramolecular interaction exhibiting remarkable quenching of fluorescence spectrum and lifetime (tau = 3.5 ns) in the absence of DNA. In the presence of DNA, however, the 3.5-ns lifetime component of fluorescence disappeared, and a two-exponential decay of fluorescence (t = approximately 10 and 1.5 ns) was observed at a DNA concentration of more than approximately 0.001 mM P, while the solution containing a very dilute DNA concentration (less than or equal to 0.001 mM P) exhibits a three-component decay of fluorescence (1.5, 3.5, and approximately 10 ns). The potent bis intercalation of two moieties in 2GP-SP with an identical DNA molecule was suggested by the DNA-concentration dependence of these fluorescence lifetimes and their intensity.

Determinação da razão ótima de espermatozóides por ovócitos de piabanha Brycon insignis (pisces - characidae) / Determination of the optimum ratio of spermatozoa per oocyte of the piabanha Brycon insignis

Determinou-se a razão ótima de espermatozóides por ovócitos da piabanha Brycon insignis, utilizando-se dois machos e duas fêmeas da espécie, submetidos ao procedimento de desova induzida. Os gametas foram extrusados manualmente 200 horas-grau após a aplicação do extrato bruto de hipófise. Os ovócitos foram misturados e deste pool retiraram-se amostras com 2g de ovócitos (701 ovócitos/g). O sêmen do pool foi diluído em solução de NaCL 1,2 por cento de tal forma que, após a adição de 1ml do sêmen diluído aos ovócitos, fossem obtidas as seguintes razões espermatozóides por ovócitos em cada tratamento: T1=86.662, T2=173.324, T3=259.986, T4=346.648 e T5=433.310. A taxa de fertilização do sêmen não diluído usado como controle foi de 65,3 por cento. Após ativação espermática com NaHCO3 1 por cento e fecundação, os ovos foram transferidos para as incubadoras e nelas foram observadas as seguintes percentagens de fertilização em relação à do grupo-controle: T1=35,7 por cento, T2=53,1 por cento, T3=79,1 por cento, T4=93,4 por cento e T5=87,8 por cento. A percentagem de fertilização (em relação ao controle) aumentou de forma linear, segundo a equação de regressão: Y=15,55+0,0002297X (P<0,01; R²=0,98), até a proporção de 314.481 espermatozóides por ovócitos, após o que a taxa de fertilização se estabilizou no limite de 88 por cento

The optimum spermatozoa:oocyte ratio of piabanha Brycon insignis was studied. Two males and two females were induced to spawn, and the gametes were stripped after 200 hourgrades starting from the application of carp pituitary gland. Oocytes from two females were mixed, and samples of 2g (701 oocytes/g) were collected from the obtained pool and placed into plastic cups. The semen from the two males, after mixed to compose a pool, was diluted in NaCl solution (1.2 percent) so that, after the addition of 1ml of diluted sperm into the oocytes, the following spermatozoa:oocyte ratios (tri-replicated) were obtained: T1=86,662, T2=173,324, T3=259,986, T4=346,648 and T5=433,310. After the activation using 1 percent NaHCO3 and fertilization, the eggs were transferred to incubators, and observed the percentages of fertilization relative to the control. T1=35.7 percent, T2=53.1 percent, T3=79.1 percent, T4=93.4 percent and T5=87.8 percent. Undiluted semen was used as "control", which showed 65 percent of fertilization. The percentage of fertilization (relative to the control) increased linearly according to the regression Y= 15.55 + 0.0002297X (P<0.01; R²=0.98), until the proportion of 314,481 spermatozoa per oocyte, and, from this point, the fertilization rate maintained at 88 percent