Assessment of DNA damage in goat preantral follicles after vitrification of the ovarian cortex.

Effective methods for gamete preservation should have low impact on DNA integrity. The present study investigated the effects of vitrification of goat ovarian tissues on the occurrence of DNA fragmentation and DNA double-stand breaks using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay and detection of phosphorylated histone H2AX (γH2AX), respectively. Goat ovaries were collected at a local abattoir and 12 tissue fragments were prepared from each ovarian pair. Tissue fragments were used as fresh control samples or were cultured in vitro, vitrified or vitrified and cultured. Vitrification was performed using the Ovarian Tissue Cryosystem. Fragments from all groups (control and treatments) were processed for histology, transmission electron microscopy, TUNEL assay and immunofluorescence. Compared with fresh control samples, a lower percentage of morphologically normal follicles was detected in the vitrification followed by culture treatment group (P<0.05). Normal follicular ultrastructure was observed in all groups. Immunofluorescence revealed the presence of γH2AX foci in few oocytes and ovarian stromal cells. TUNEL-positive follicles were found in samples without significant differences among groups (P>0.05). In conclusion, the vitrification protocol used in the present study did not increase DNA damage in preantral follicles enclosed in goat ovarian tissues.

Steady-state level of epidermal growth factor (EGF) mRNA and effect of EGF on in vitro culture of caprine preantral follicles.

Our aim was to verify the steady-state level of epidermal growth factor (EGF) mRNA in goat follicles at various developmental stages and to investigate the influence of EGF on the survival, antrum formation and growth of secondary follicles cultured for 6 days. Primordial, primary and secondary goat follicles and small and large antral follicles were obtained to quantify EGF mRNA by real-time reverse transcription with the polymerase chain reaction. The influence of EGF and the presence or absence of follicle-stimulating hormone (FSH) on the development of secondary follicles and on mRNA expression for EGF and FSH receptor (FSH-R) was determined after 6 days of culture. Survival, antrum formation and follicular diameter were evaluated every other day of culture. EGF mRNA levels in secondary follicles were significantly higher than those in primordial follicles, whereas in small and large antral follicles, EGF mRNA levels in cumulus-oocyte complexes (COCs) were significantly higher than in granulosa/theca cells. During culture, EGF in the presence or absence of FSH increased the follicular daily growth rate of secondary follicles when compared with that in enriched alpha minimal essential medium. FSH, EGF or both reduced EGF mRNA levels, whereas EGF reduced FSH-R mRNA levels after follicle culture for 6 days. Thus, EGF mRNA levels are higher in secondary follicles than in earlier stages, with both FSH and EGF promoting the growth of goat secondary follicles. EGF and/or FSH reduce EGF mRNA levels, whereas EGF decreases FSH-R mRNA levels, in cultured secondary follicles.

In Vitro Activation and Development of Goat Preantral Follicles Enclosed in Ovarian Tissue Co-cultured with Mesenchymal Stem Cells.

The development of culture systems capable of maintaining follicular growth since the preantral stage has been the target of investigations. Mesenchymal stem cells (MSC) present potential for use in a wide range of applications, including research aimed at preserving fertility. Therefore, this study investigated the use of caprine Wharton's Jelly Mesenchymal Stem Cells (WJMSC) on the survival and in vitro development of goat preantral follicles enclosed in ovarian fragments cultured for 1 or 7 days. Fragments of the ovarian cortex were immediately fixed (non-cultured control) or distributed in four treatments: ovarian tissue cultured in control medium (α-MEM+); ovarian tissue cultured in α-MEM+ supplemented with FBS (α-MEM+ + FBS); ovarian tissue co-cultured with stem cells in α-MEM+ (α-MEM+ + SC); and ovarian tissue co-cultured with stem cell in α-MEM+ + FBS (α-MEM+ + SC + FBS). The rates of cell proliferation, follicular survival, and activation, as well as follicular diameter, were evaluated. After 7 days, the treatment co-cultured with stem cells showed a higher (P < 0.05) percentage of morphologically normal preantral follicles compared to the other treatments, as well as a higher (P < 0.05) activation rate compared to cultured control. Moreover, the follicular diameter was higher (P < 0.05) in the treatment co-cultured with stem cells compared to co-cultured with stem cells plus FBS. This study demonstrates for the first time that in vitro co-culture of caprine WJMSC with preantral follicles enclosed in goat ovarian tissue improves activation and early follicular development.

Response of vital functions, Apgar and cortisol in the prognosis of vigor against neonatal factors of lambs / Resposta das funções vitais, de Apgar e cortisol no prognóstico de vigor frente a fatores neonatais de cordeiros

The management of lambs during the neonatal period has been studied in several researches due to the vital and hormonal organic adaptations undergone by the calf after birth. However, gender, number of pups and type of delivery play an important role in understanding neonatal vigor. The study of these groups with the monitoring of clinical evolution and cortisol metabolism becomes an indispensable subsidy for a better understanding of this neonatal phase, aiming to minimize the losses generated. The objective of this study was to evaluate the influence of gender, number of pups and type of delivery in the prognosis of neonatal vigor of lambs through clinical and cortisol diagnosis. Thirty crossbred Santa Inês lambs with Dorper in the neonatal phase were divided into three groups: male and female, number of pups (single and twin) and type of delivery (eutocic and dystocic). In each group, clinical evaluation of heart and respiratory rate, rectal temperature, Apgar score and weight were performed; and with the exception of cortisol, all evaluations were performed at fifteen and sixty minutes, as well as at twelve and twenty-four hours. In addition, blood samples were collected for cortisol dosage obtained in two moments at fifteen and sixty minutes using the radioimmunoassay technique. Among the three experimental groups related to lamb vigor, the heart rate was the only one that showed lower mean values (P<0.05) at twenty-four hours in the male group 90.00±20.20bpm, twins 96.44±20.02bpm and eutocic 93.25±18.11bpm. Differences in respiratory rate values were observed in the eutocic group (64.00±14.75mpm) at twenty-four hours. In the group of males there was a significant reduction in body temperature during the evaluation moments (P<0.05). Lambs from the group of twins showed lower body weight during the evaluations. At both times the analysis of serum cortisol was less than at sixty minutes. It was concluded that soon after the birth there were marked changes in the physiological parameters and weight of Santa Inês lambs, but were not enough to cause negative effects on the vigor of the neonates, indicating the occurrence of effective neonatal adaptation capacity in this species.(AU)

O manejo dos cordeiros durante o período neonatal tem sido objeto de estudo em diversas pesquisas devido às adaptações orgânicas vitais e hormonais sofridas pela cria após o parto. Todavia, o gênero, número de filhotes e o tipo de parto parecem desempenhar um papel importante para melhor compreensão do vigor neonatal. Além disso, o estudo destes grupos com o acompanhamento da evolução clínica e do metabolismo do cortisol torna-se um subsidio indispensável para melhor compreensão dessa fase neonatal, visando minimizar as perdas geradas. Dessa forma, o objetivo do presente trabalho foi avaliar a influência do gênero, número de filhotes e tipo de parto na apresentação do vigor neonatal dos cordeiros através do diagnostico clinico e de cortisol. Foram utilizados trinta cordeiros mestiços da raça Santa Inês com Dorper em fase neonatal divididos em três grupos: gênero (macho e fêmea), número de filhotes (único e gemelar) e tipo de parto (eutócico e distócico). Em cada grupo, foi realizada a avaliação clínica da frequência cardíaca e respiratória, temperatura retal, escore Apgar e peso; e com a exceção do cortisol, todas as avaliações foram realizadas aos quinze e sessenta minutos, como também às doze e vinte e quatro horas. Adicionalmente, procedeu-se com a coleta de amostras de sangue total para dosagem de cortisol obtida em dois momentos aos quinze e sessenta minutos através da técnica de radioimunoensaio. Dentre os três grupos experimentais relacionados com vigor dos cordeiros, a frequência cardíaca foi a única que evidenciou menores médias (P<0,05) às vinte e quatro horas no grupo dos machos 90,00±20,20bpm, gêmeos 96,44±20,02bpm e eutócicos 93,25±18,11bpm. Observou-se no grupo eutócico diferenças nos valores da frequência respiratória de 64,00±14,75mpm às vinte e quatro horas. No grupo dos machos houve redução significativa na temperatura corpórea durante os momentos de avaliação (P<0,05). Cordeiros do grupo de gêmeos demonstraram menor peso corpóreo durante as avaliações. Em ambos momentos a análise do cortisol sérico demonstrou se menor aos sessenta minutos. Pôde se concluir que logo após o parto ocorreram alterações marcantes nos parâmetros fisiológicos e peso de cordeiros Santa Inês, porém não foram suficientes para causar efeitos negativos sobre o vigor dos neonatos, indicando a ocorrência de efetiva capacidade de adaptação neonatal nesta espécie.(AU)

Catalase prevents lipid peroxidation and enhances survival of caprine preantral follicles cryopreserved in a 1,2-propanediol-freezing medium.

The objectives of this study were to determine: 1) the optimal concentration (1.0 or 1.5 M) and duration of exposure (5, 10, or 20 min) of ovarian tissue to 1,2-propanediol (PROH) on morphology and viability of caprine preantral follicles; and 2) the effect of supplementing cryopreservation medium supplementation with Trolox(®) (0.1, 0.5, or 1.0 mM) or catalase (5, 10, or 20 IU/mL) on follicular morphology, viability, and lipid peroxidation. Cryopreservation decreased (p<0.05) percentages of normal follicles relative to the control (84%). Although supplementation of the cryopreservation medium (1.0 M PROH) with catalase (10 or 20 IU/mL) or Trolox(®) (0.1 mM) resulted in follicular morphology and viability similar to that in the controls (P>0.05), lipid peroxidation was reduced only when 20 IU/mL catalase was added to the cryopreservation medium.

Quantification of dimethyl sulfoxide perfusion in sheep ovarian tissue: a predictive parameter for follicular survival to cryopreservation.

The aim of the present study was to determine the amount of dimethyl sulfoxide (DMSO) present in sheep ovarian tissue after exposure to cryoprotectant at different times (5, 10, 20, or 30 min) and at different concentrations (1.0, 1.5, or 2.0 M). To quantify the levels of DMSO in the ovarian tissue, the high-performance liquid chromatography (HPLC) method was applied. In addition, viability of preantral follicles after toxicity test and cryopreservation of ovarian tissue using the above mentioned concentrations of DMSO and exposure times was evaluated. We have observed that the presence of ∼0.6 mg of DMSO into the ovarian tissue may be deleterious to the sheep preantral follicles. In addition, the application of a short exposure time (5 min at 1.5 or 2.0 M DMSO) or low concentration (1.0 M for 10 min) of DMSO successfully preserves sheep preantral follicles following cryopreservation.