Effects of Two Types of Melatonin-Loaded Nanocapsules with Distinct Supramolecular Structures: Polymeric (NC) and Lipid-Core Nanocapsules (LNC) on Bovine Embryo Culture Model.

Melatonin has been used as a supplement in culture medium to improve the efficiency of in vitro produced mammalian embryos. Through its ability to scavenge toxic oxygen derivatives and regulate cellular mRNA levels for antioxidant enzymes, this molecule has been shown to play a protective role against damage by free radicals, to which in vitro cultured embryos are exposed during early development. In vivo and in vitro studies have been performed showing that the use of nanocapsules as active substances carriers increases stability, bioavailability and biodistribution of drugs, such as melatonin, to the cells and tissues, improving their antioxidant properties. These properties can be modulated through the manipulation of formula composition, especially in relation to the supramolecular structures of the nanocapsule core and the surface area that greatly influences drug release mechanisms in biological environments. This study aimed to evaluate the effects of two types of melatonin-loaded nanocapsules with distinct supramolecular structures, polymeric (NC) and lipid-core (LNC) nanocapsules, on in vitro cultured bovine embryos. Embryonic development, apoptosis, reactive oxygen species (ROS) production, and mRNA levels of genes involved in cell apoptosis, ROS and cell pluripotency were evaluated after supplementation of culture medium with non-encapsulated melatonin (Mel), melatonin-loaded polymeric nanocapsules (Mel-NC) and melatonin-loaded lipid-core nanocapsules (Mel-LNC) at 10-6, 10-9, and 10-12 M drug concentrations. The highest hatching rate was observed in embryos treated with 10-9 M Mel-LNC. When compared to Mel and Mel-NC treatments at the same concentration (10-9 M), Mel-LNC increased embryo cell number, decreased cell apoptosis and ROS levels, down-regulated mRNA levels of BAX, CASP3, and SHC1 genes, and up-regulated mRNA levels of CAT and SOD2 genes. These findings indicate that nanoencapsulation with LNC increases the protective effects of melatonin against oxidative stress and cell apoptosis during in vitro embryo culture in bovine species.

Vitrification of equine oocytes with a polyvinyl alcohol after in vitro maturation with equine growth hormone and insulin-like growth factor-I.

BACKGROUND: In vitro fertilization (IVF) procedures are limited by the inability to mature equine oocytes on in vitro methods. OBJECTIVE: The aim of this study was to evaluate structural integrity of equine oocytes subjected to vitrification with a synthetic polymer (PVA). METHODS: The effect of eGH and its relationship with IGF-I on in vitro maturation (IVM) were evaluated. Compact cumulus oocytes complexes (n=122) were cultured in TCM-199 with eGH, IGF-I or eGH+IGF-I for 30h at 38.5C in air with 5 % CO2. Oocytes were fixed after IVM or subjected to the vitrification protocol. Cryopreserved oocytes were exposed to 1.4M DMSO+1.8M EG+1 percent PVA for 3min, and then transfer to 2.8M DMSO+3.6M EG+0.6M sucrose+1 % PVA for 1min. After rewarming, oocytes were evaluated by confocal microscopy. RESULTS: Maturation rates of oocytes were not significant different among groups (P > 0.05), however eGH+IGF-I group can develop the assessment of resumption of meiosis (MI+MII = 86.7 %). CONCLUSION: The oocyte did not show morphological alterations. The use of PVA-copolymer may represent a potential alternative for vitrification of equine oocytes after IVM.

Binding pattern and toxicological effects of lectins from genus Canavalia on bovine sperm.

The aim of this study was to determine the binding patterns of Canavalia ensiformis (ConA), Canavalia boliviana (ConBol) and Canavalia brasiliensis (ConBr) lectins to bovine sperm and their effects on sperm motility, viability, lipid peroxidation, reactive oxygen species production and fertilization ability. ConA bound to whole spermatozoa, with the exception of the equatorial segment, ConBol did not interact with the acrosome region and ConBr exhibited a fragmented binding pattern. The three lectins decreased sperm motility but did not affect cell viability or lipid peroxidation. Nevertheless, ROS production was increased in comparison to controls and a reduction in the cleavage and blastocyst ratio was induced in comparison to controls. In conclusion, this study determined that structurally similar lectins interact differently with bovine sperm and affect sperm motility, viability, lipid peroxidation, ROS production and fertilization ability in various ways.

Association between single nucleotide polymorphisms in p53 and abortion in Thoroughbred mares.

Single nucleotide polymorphisms (SNPs) in the p53 gene have been studied extensively in humans. The aims of this study were to determine the frequency of the Arg/Pro SNP in p53 in Thoroughbred mares on one stud in Brazil and to correlate p53 genotypes with reproductive performance. SNPs were detected by PCR-restriction fragment length polymorphism in blood samples from 105 horses and confirmed by sequencing. The allele frequency in Thoroughbred mares at codon 72 in exon 4 was 73.3% Arg/Pro, 17.1% Arg/Arg and 9.6% Pro/Pro. The presence of Arg/Pro was significantly associated with abortion (P=0.02), while Pro/Pro mares had a lower probability of abortion (P<0.05). Using a logistic regression model, the dominance effect was significant (P=0.044; odds ratio 7.94) for abortion and additive effects were not significant (P=0.26). p53 may play a role in equine reproduction.

Equine fetal sex determination using circulating cell-free fetal DNA (ccffDNA).

In this study, polymerase chain reaction (PCR) reamplification of the first PCR product (2nd-PCR) and a qPCR assay were used to detect the sex determining region Y (SRY) gene from circulating cell-free fetal DNA (ccffDNA) in blood plasma of pregnant mares to determine fetal sex. The ccffDNA was isolated from plasma of 20 Thoroughbred mares (5-13 y old) in the final 3 mo of pregnancy (fetal sex was verified after foaling). For controls, plasma from two non-pregnant mares and two virgin mares were used, in addition to the non-template control. The 182 bp nucleotide sequence corresponding to the SRY-PCR product was confirmed by DNA sequencing. Based on SRY/PCR, 8 of 11 male and 9 of 9 female fetuses were correctly identified, resulting in a sensitivity of 72.7% (for male fetuses) and an overall accuracy of 85%. Furthermore, using SRY/2nd-PCR and qPCR techniques, sensitivity and accuracy were 90.9 and 95%, respectively. In conclusion, this study is apparently the first report of fetal sex determination in mares using ccffDNA.

NPY and sbGnRH gene expression in juvenile and adult male Brazilian flounder Paralichthys orbignyanus / Expressão gênica do NPY e do sbGnRH em machos juvenis e adultos de linguado Paralichthys orbignyanus

The objective of this study was to evaluate neuropeptide Y (NPY) and sea bream gonadotropin-release hormone (sbGnRH) gene expression in juvenile and adult males of Brazilian flounder. Hypothalamuses from fish were sampled for total RNA extraction. After cDNA synthesis, real-time PCR was used to measure gene expression. NPY showed approximately 2-fold increases in their mRNA levels while sbGnRH showed 3-fold increases in adult fish. These results suggest that these peptides could be involved on hypothalamic regulation of Brazilian flounder sexual maturation.

O objetivo deste estudo foi avaliar a expressão gênica do neuropeptídeo Y (NPY) e da variante sea bream do hormônio liberador de gonadotrofinas (sbGnRH) em linguados machos juvenis e adultos. O hipotálamo foi isolado para a extração de RNA total. Após a síntese de cDNA, a PCR em tempo real foi usada para avaliar a expressão gênica. Foi observado um aumento de aproximadamente duas vezes nos níveis de NPY e de aproximadamente três vezes nos níveis de sbGnRH nos peixes adultos. Esses resultados demonstram que estes peptídeos podem estar envolvidos na regulação, via hipotálamo, da maturação sexual no linguado.

Transgene transmission in chickens by sperm-mediated gene transfer after seminal plasma removal and exogenous DNA treated with dimethylsulfoxide or N,N-dimethylacetamide.

Transgenic animals have been successfully produced by mass gene transfer techniques such as sperm-mediated gene transfer (SMGT). The aim of this work was to demonstrate transgene transmission by SMGT in chickens using dimethylsulfoxide (DMSO) or N,N-dimethylacetamide (DMAc) as transfectants after seminal plasma removal to prevent DNase activity. Sperm samples were prepared by repetitive washes, and after each wash sperm motility, seminal plasma proteins, exogenous DNA integrity and its uptake by spermatozoa were evaluated. Laying hens were inseminated using spermatozoa transfected with pEGFP-N1 vector in the presence of DMSO or DMAc. Transgene transmission in newborn chicks was evaluated by in vivo enhanced green fluorescent protein (EGFP) expression, RT-PCR and PCR analysis. DNA internalization was limited to sperm samples washed twice. The presence of DMSO or DMAc during transfection had no effect on fertilization or hatching rates. PCR analysis detected the presence of EGFP DNA in 38% of newborn chicks from the DMSO group and 19% from the DMAc group. EGFP mRNA was detected in 21% of newborn chicks from the DMSO group, as against 8.5% from the DMAc group. However, in vivo expression of EGFP was only observed in a single animal from the DMSO group. Our data revealed that the plasmid DNA-DMSO combination coupled with sperm washes can be an efficient method for transfection in chickens.

NanoSMGT: transgene transmission into bovine embryos using halloysite clay nanotubes or nanopolymer to improve transfection efficiency.

The objectives were to investigate whether: 1) nanotransfectants are more effective than other common transfection methods for SMGT; 2) NanoSMGT is able to transmit exogenous DNA molecules to bovine embryos; and 3) halloysite clay nanotubes (HCNs) can be used as a transfection reagent to improve transgene transmission. Four transfection systems were used: naked DNA (without transfectant), lipofection, nanopolymer, and halloysite clay nanotubes. Plasmid uptake by sperm and its transfer to embryos were quantified by conventional and real-time PCR, as well as EGFP expression by fluorescence microscopy. Furthermore, sperm motility and viability, and embryo development were investigated. Mean number of plasmids taken up was affected (P < 0.05) by transfection procedure, with the nanopolymer being the most effective transfectant (∼ 153 plasmids per spermatozoon). None of the treatments affected sperm motility or viability. The mean number of plasmids transmitted to four-cell stage embryos was higher (P < 0.05) in nanopolymer and HCNs than liposomes and naked DNA groups. The number of embryos carrying the transgene increased from 8-10% using naked DNA or liposomes to 40-45% using nanopolymer or HCN as transfectants (P < 0.05). There were no significant differences among transfection procedures regarding blastocyst formation rate of resulting embryos. However, no EGFP-expressing embryo was identified in any treatment. Therefore, nanotransfectants improved transgene transmission in bovine embryos without deleterious effects on embryo development. To our knowledge, this was the first time that bovine embryos carrying a transgene were produced by NanoSMGT.

MicroRNAs e seu papel no desenvolvimento embrionário / MicroRNAs and its role in embryonic development

MicroRNAs (miRNAs) são pequenas moléculas de RNA com aproximadamente 22 nucleotídeos incapazes de codificar proteínas e que apresentam função na regulação pós-transcricional da expressão gênica. Vários estudos vêm demonstrando o importante papel dos miRNAs na regulação do desenvolvimento embrionário de diferentes espécies, desde o controle da expressão de RNAs mensageiros durante o desenvolvimento inicial embrionário até a determinação de linhagens celulares durante a organogênese. Esta revisão irá abordar os principais miRNAs e seu papel na biologia reprodutiva, com ênfase no desenvolvimento embrionário de mamíferos.

MicroRNAs (miRNAs) are small RNA molecules with around 22 nucleotides that are unable to encode proteins and play a key role on post-transcription regulation process. Several studies have demonstrated the relevant role of miRNAs on the regulation of embryonic development on different species, from the control of gene expression during the early embryo development to determination of cellular lineages over the organogenesis. This review will present the miRNAs and its role on reproductive biology focusing on the mammalian embryo development.

Transgene transmission in South American catfish (Rhamdia quelen) larvae by sperm-mediated gene transfer.

The silver catfish (Rhamdia quelen) is an endemic American fish species. The sperm of each species has its own peculiarities and biological characteristics, which influence the success of mass DNA transfer methods. Our objective in this study was to evaluate different sperm-mediated gene transfer (SMGT) methods to obtain transgenic silver catfish. Different treatments for the incorporation of a foreign pEGFP plasmid group were used: (1) dehydrated/rehydrated (DR), (2) dehydrated/rehydrated/electroporated (DRE), (3) electroporated (E), (4) incubated with seminal plasma (INC); and (5) incubated in the absence of seminal plasma (INCSP). Sperm motility, time of activity duration (TAD), fertilization rate (FR), hatching rate (HR) and sperm morphology were also evaluated. The polymerase chain reaction (PCR) positivity rates for the presence of the transgene were: DRE 60%; DR 40%; E 25%; INC 5% and INCSP 25%. The rates of embryo EGFP expression were: DRE 63%; DR 44%; E 34%; INC 8% and INCSP 38%. The fertilization rate in the control and DRE treatments groups were higher than in the DR group, but the E,INC and INCSP treatment groups had the lowest rate. The hatching rates of the DRE, DR and control groups were higher than in the INCSP, INC and E treatment groups (P>0.05). There were no differences among the DRE and DR, E and DR, E and INCSP groups in expression and PCR positivity rates of enhanced green fluorescent protein (EGFP) in embryos. Scanning electron microscopy also did not show any change in sperm morphology among treatment groups. To the best of our knowledge, this is the first report on transgene transmission of exogenous DNA into silver catfish larvae through SMGT technology.

Inseminação artificial intra-uterina em leitoas com sêmen criopreservado com dimetilacetamida e glicerol / Intrauterine artificial insemination of gilts using cryopreserved boar semen with dimetilacetamide and glycerol

Este estudo teve por objetivo avaliar o uso da dimetilacetamida (DMA) e de glicerol na criopreservação de sêmen suíno sobre as taxas de concepção e fertilização in vivo, utilizando o método de inseminação artificial pós-cervical. Foram sincronizadas 60 leitoas pré-púberes e inseminadas com o uso de sêmen congelado com glicerol 3 por cento (30 fêmeas) e DMA 5 por cento (30 fêmeas). O método de inseminação utilizado foi o pós-cervical, com concentração de 1 x 10(9) espermatozóides vivos por dose. Após 36 a 40h da inseminação, as fêmeas foram abatidas, sendo realizada a contagem de corpos hemorrágicos (CH) nos ovários. Foi realizada a lavagem dos ovidutos das fêmeas, verificando o número de estruturas recuperadas (oócitos e embriões), calculando-se as taxas de concepção e fertilização. A média de CH nas fêmeas do grupo glicerol 3 por cento não diferiu (P>0,05) daquelas do grupo DMA 5 por cento (10,4 x 10,2, respectivamente). Não houve diferença (P>0,05) nas taxas de recuperação de estruturas entre os grupos glicerol 3 por cento (68,9 por cento) e DMA 5 por cento (66,9 por cento). Os resultados obtidos nos grupos glicerol 3 por cento e DMA 5 por cento para as taxas de concepção (73,3 x 76,6 por cento) e fertilização (48,6 x 59,4 por cento) não apresentaram diferença (P>0,05). Conclui-se que não há diferenças nas taxas de concepção e fertilização in vivo utilizando-se sêmen congelado com o uso de dimetilacetamida ou de glicerol.

The objective of this study was to evaluate the use of dimethylacetamide (DMA) and glycerol in boar semen cryopreservation on the conception and fertility rates.Sixty pre-pubertal gilts were synchronized and inseminated with semen including either glycerol 3 percent or DMA 5 percent as cryoprotectants (n=30 for both groups). Artificial insemination was conducted with the intrauterine method using doses with 1 x 10(9) viable spermatozoa. The gilts were slaughtered 36-40h after the insemination and the hemorrhagic bodies (CH) in the ovaries were counted. The oviducts were washed, so oocytes and embryos could be recovered to determine the conception and fertility rates. The average CH obtained with glycerol 3 percent did not differ (P>0.05) from those observed with DMA (10.4 x 10.2, respectively). Recovery rates also did not differ (P>0.05) between groups (68.9 percent with glycerol and 66.9 percent with DMA). Conception (73.3 x 76.6 percent) and fertility rate (48.6 x 59.4 percent) were also similar (P>0.05) for glycerol and DMA, respectively. Those results indicate that DMA 5 percent can replace glycerol 3 percent in the composition of extenders for frozen boar semen.

Proporção macho: fêmea de embriões bovinos cultivados na presença ou ausência de glicose após FIV com espermatozóides selecionados por Swim-up ou Gradiente de Percoll

No sistema de PIV em bovinos, tem sido obtida uma elevada porcentagem de embriões machos. Este experimento foi realizado para determinar se a presença de glicose no meio de cultivo afeta a proporção macho:fêmea (M:F) dos embriões bovinos PIV a partir da FIV com espermatozóides preparados pelos métodos do swim-up (S) ou do gradiente de Percoll (P). Após a MIV, os COCs foram divididos em dois grupos e inseminados com espermatozóides preparados por um dos métodos. Os zigotos foram cultivados em meio com ou sem 5,56mM de glicose, totalizando 4 tratamentos: S-Gli, S+Gli, P-Gli e P+Gli e 48h após a inseminação, os embriões de cada tratamento foram submetidos à sexagem por PCR (n=845). O efeito da glicose no meio de cultivo sobre a proporção M:F dos embriões PIV a partir dos dois métodos foi semelhante (teste do c²), resultando em uma porcentagem de machos menor do que 50 por cento no estágio de 2-C (S: 30,8 por cento; P: 23,8 por cento: P<0,01) e maior do que 50 por cento no estágio de 8-C (S: 79,4 por cento; P: 68,8 por cento: P<0,01). Estas porcentagens foram diferentes (P<0,05) das observadas quando os embriões foram cultivados sem glicose, tanto no estágio de 2-C (S: 48,5 por cento; P: 41,5 por cento) como no de 8-C (S: 62,5 por cento; P: 50,8 por cento). A presença de glicose não afetou a proporção M:F no total de embriões produzidos (S: 56,7 por cento; P: 49,0 por cento), que foi semelhante à observada na ausência de glicose (S: 55,7 por cento; P: 46,2 por cento). Portanto, a glicose exacerbou a diferença na velocidade de desenvolvimento entre os embriões machos e fêmeas.

Influencia do período de coleta sobre o volume, motilidade e doses de sêmen em suínos / Influence of the collection period on volume, motility and semen doses in swine

O presente trabalho teve como objetivo determinar ainfluência do ano e mês de coleta sobre o volume (VOL), motilidade (MOT) e doses de sêmen produzidas (DO), as correlações existentes entre as variáveis e as suas repetibilidades. Foram analisadas amostras de sêmen de 96 machos pertencentes as raças Landrace (41), Large White (31) e Duroc (24), durante o período (1981 a 1987) de permanência dos mesmos na Central de Inseminação Artificial de Suínos de Estrela - RS. O número de amostras dês sêmen coletadas foi de 7.264 da raça Landrace, 3.589 da raça Large White e 3.051 da raça Duroc. Os resultados mostraram haver influência (P<0,01) do ano e mês sobre as variáveis analisadas. Os valores médios mínimos e máximos, dentro de cada raça, respectivamente, foram: VOL de 236,9ml e 300,4ml na raça Landrace, 238, 1 ml e 284,1 ml na raça Large White e 150ml e 201.1ml na raça Duroc; MOT de 79,2% e 80,3% na raça Landrace, 76,7% e 78,0% na raça Large White e 7 7,8% e 7 9,1% na raça Duroc; DO 12,0 e 14,7 na raça Landrace, 10,1 e 13,0 na raça Large White e 9,1 e 11,9 na raça Duroc. As correlações entre VOL e DO foram as mais altas (Landrace 0,30, Large White 0,36 e Duroc 0,36). As correlações entre VOL e MOT foram próximas de zero (Landrace -0,05, Large White 0,03 e Duroc 0,01) e entre MOT e DO foram de 0,08 (Landrace}, 0,15 (Large White), e 0,13 (Duroc). A repetibilidade foi alta para VOL (Landrace 0,49, Large White 0,59 e Duroc 0,54). As outras repetibilidades foram: Landrace 0,18 para MOT e 0,30 para DO, Large White 0.27 para MOT e DO e, Duroc 0,21 para MOT e 0,39 para DO.

The aim of this experiment was to determine the influence of the collection period on volume (VOL), motility (MOT) and semen doses (DO), and the correlation among the six variables and their repeatibilities. Semen samples from ninety (96) boars belonging to Landrace (41), Large White (31) and Duroc (24) breeds were analyzed, taken into account the permanence period (1981 to 1987) of the boars at the Artificial Insemination Center -Estrela - RS. The number of semen samples collected were: Landrace 7,264, Large White 3,589 and Duroc, 3,051. Year and month of collection had influence (P<0.01) on the variables analyzed. Minimum and maximum average values, within each breed, were VOL 236.9 and 300.4ml (Landrace), 238.1 and 284.1ml (Large White) and 150.0 and 201.1ml (Duroc): MOT 79.2 and 80.3% (Landrace), 76.7 and 78.0% (Large White) and 77.8 and 79.1% (Duroc); DO 12.0 and 14.7 (Landrace), 10.1 and 13.0 (Large White) and 9.1 and 11.9 (Duroc), respectively. Correlations between VOL and DO were 0.30 (Landrace), 0.36 (Large White) and 0.36 (Duroc). Correlations between VOL and MOT were close to zero (Landrace -0.05, Large White 0.03 and Duroc 0.01), and between MOT and DO were 0.08 (Landrace), 0.15 (Large White) and 0.13 (Duroc). Repeatibilities were VOL 0.49 (Landrace), 0.59 (Large White) and 0.54 (Duroc); MOT 0.18 (Landrace), 0.27 (Large White) and 0.21 (Duroc), and DO 0.30 (Landrace), 0.27 (Large White) and 0.39 (Duroc).

Efeitos de produtos anabolizantes sobre a qualidade do sêmen de touros / Effects of anabolic products on bull semen quality

Com a finalidade de se avaliar o efeito de esteróides anabolizantes sobre a qualidade do sêmen de touros, foram testados 3 tratamentos em 9 touros adultos (idades de 3 a 5 anos). Os grupos I e II receberam implantes subcutâneos na base da orelha, sendo implantados no grupo I, 36mg de zeranol e no grupo II 24mg de dietiletilbestrol (DES), enquanto o grupo III permaneceu como testemunha. Foram realizadas 31 coletas de sêmen semanais, tendo sido a primeira, uma semana antes do implante. Não se verificaram alterações em nenhuma das características do sêmen devido aos tratamentos. Os três grupos tiveram ganhos de peso similares. Conclui-se que, nas condições em que o experimento foi realizado, não houve efeito dos anabolizantes na qualidade do sêmen. Caso tenham ocorrido alterações genitais mais discretas, essas foram subclínicas ou não foram detectadas devido ao número de unidades experimentais por tratamento.

In order to evaluate the effect of anabolic steroids on the semen quality of bulls, 3 treatments were tested in 9 adult bulls (3 to 5 years old). Groups I and II received subcutaneous implants in the base of the ear while group III remained as control. bulls from group I were treated with 36mg of Zeranol and group II 24mg of diethylethylbestrol (DES). A total of 31 weekly semen collections were performed, being the first, one week before the implant. No changes on semen characteristics were observed due to treatments. The 3 groups had similar weight gain. It was concluded that, in the conditions of mis experiment, there was no effect of the anabolic products on semen quality. In case that some mild genital changes had ocurred, they were subclinical or were not detected due to the number of experimental units per treatment.

Efeito da concentração e temperatura de adição de trealose em diluentes para a congelação de sêmen ovino em palhetas / Effect of the concentration and addition temperature of trehalose in extenders on freezing ram semen in straws

A trealose é um dissacarídio com potencial emprego como crioprotetor quando adicionada aos meios para congelamento de sêmen ovino. Este experimento foi realizado para verificar os efeitos da temperatura de adição (30°C e 4°C) e da concentração de trealose (2 por cento, 4 por cento e 6 por cento) sobre o sêmen ovino congelado em palhetas, utilizando como base as formulações INRA e TRIS/FRUTOSE. Os efeitos estudados em ambos experimentos foram medidos através da avaliação da motilidade espermática (MOT) e da integridade de acrossomas (INTA) em diferentes momentos após o descongelamento (0h, 2h e 5h). Os presentes resultados não recomendam a inclusão da trealose visando incrementar a qualidade in vitro do sêmen ovino congelado em palhetas nas concentrações e diluentes testados, porém, sugerem maiores estudos quanto a sua toxidade e possíveis interações com outros constituintes dos diluentes já formulados para o congelamento de sêmen ovino.

This study was aimed to evaluate the possible effects of the addition of trehalose to extenders developed for freezing ram semen in straws. The effects of addition temperature (30°C and 4°C) and concentration of trehalose (2, 4 and 6 percent) on INRA and TRIS/FRUTOSE diluents was evaluated. Their effects were studied through motility rate and by acrosome integrity at different incubation times after thawing (0, 2 and 5h). The results do not recommend the inclusion of trehalose in these diluents. However, it would be interesting to learn more about toxicity and interactions between the components of the extenders and trehalose in ram sperm frozen in straws.

Gonadotrofina coriônica eqüina: purificação, caracterização e resposta ovariana em ovinos e suínos / Equine chorionic gonadotrophin: purification, characterization and ovarian activity in ewes and gilts

A gonadotrofina coriônica equina (eCG) foi purificada e caracterizada com respeito ao grau de pureza e atividade biológica. A pureza de quatro preparações foi determinada por eletroforese e a atividade biológica pelo incremento do peso ovariano de ratas imaturas (40 - 50g) e pela indução de ovulação em ovelhas e leitoas. A análise eletroforética revelou a presença de três bandas polipeptídicas. A atividade biológica media foi de 313 UI/mg de proteína. Sessenta e cinco (65) ovelhas, fora da estação reprodutiva, foram divididas ao acaso em dois grupos os quais receberam implantes vaginais de esponjas impregnadas com acetato de medroxiprogesterona por um período de l l a 14 dias. No grupo I (55 ovelhas), foram injetadas (IM) 500UI do eCG purificado no momento da retirada das esponjas, enquanto que no grupo II (10 ovelhas) foram injetadas 500UI de eCG comercial. Uma semana após a aplicação do eCG as ovelhas foram submetidas a um exame laparoscópico para avaliar o número de ovulações. Obteve-se uma média de 2,1 ± 0,3 e 1,8 ± 0,3 ovulações (P>0,05) para as ovelhas dos grupos I e II, respectivamente. De 120 leitoas pré-púberes, com peso médio de 87,2 kg, 90 (grupo I) foram injetadas com 500UI do eCG purificado e, às 72 horas, 500UI de hCG (gonadotrofina coriônica humana), e 30 leitoas (grupo II) não receberam injeção hormonal. Observou-se a presença de 25,9 ± 22,2 e 0,0 corpora lutea (P<0,001), nos grupos I e II, respectivamente. Estes resultados demonstraram que o PMSG purificado apresenta pureza e atividade biológica similares ao produto comercial utilizado como controle e que é eficiente para induzir atividade ovariana em ovinos e suínos.

Equine chorionic gonadotrophin (eCG) was purified and characterized with respect to its purity and bionological activity. The purity of four preparations was determined by electrophoresis, and the biological activity by increasing of the ovarian weight ot immature female rats (40-50g) and induction ot ovulation of ewes and gilts. Electrophoretic analysis revealed three polipeptidic bands. The mean biological activity was 313UI/mg of protein. Sixty-five ewes, not in reproductive season, were divided randomly in two groups that received vaginal pessaries impregnated with medroxiprogesterone acetato for 11 to 14 days. In treatment I, ewes (n = 55) were injected (IM) with 500UI of puritied eCG at the moment of pessaries withdraw, while in treatment II, the ewes (n = 10) received 500UI of comercial eCG. The results observed were 2.1 ± 0.3 and 1.8 ± 0.3 ovulations (P > 0.05) for treatments I and II, respectively. One hundred-twenty gilts, with mean weight of 87.2kg, were divided m two treatments. The animals in treatment I (90 gilts) received 500UI of puntied eCG and, 72 hours later, 500UI of hCG (human chorionic gonadotrophin). In treatment II hormones were not injected. The results observed were 25.9 ± 22.2 and 0.0 corpora lutea (P < 0.001) for treatments I and II, respectively. These results demonstrated that the eCG purified has purity and biological activity similar to the comercial product used as control, and that it is efficient in inducing ovarian activity in ewes and gilts.